1B20 PCSK9 antagonists

ABSTRACT

Antagonists of human proprotein convertase subtilisin-kexin type 9 (“PCSK9”) are disclosed. The disclosed antagonists are effective in the inhibition of PCSK9 function and, accordingly, present desirable antagonists for use in the treatment of conditions associated with PCSK9 activity. The present invention also discloses nucleic acid encoding said antagonists, vectors, host cells, and compositions comprising the antagonists. Methods of making PCSK9-specific antagonists as well as methods of using the antagonists for inhibiting or antagonizing PCSK9 function are also disclosed and form important additional aspects of the present disclosure.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application No.61/063,980, filed Feb. 7, 2008.

STATEMENT REGARDING FEDERALLY-SPONSORED R&D

Not Applicable.

REFERENCE TO MICROFICHE APPENDIX

Not Applicable.

BACKGROUND OF THE INVENTION

Proprotein convertase subtilisin-kexin type 9 (hereinafter called“PCSK9”), also known as neural apoptosis-regulated convertase 1(“NARC-1”), is a proteinase K-like subtilase identified as the 9^(th)member of the secretory subtilase family; see Seidah et al., 2003 PNAS100:928-933. The gene for PCSK9 localizes to human chromosome1p33-p34.3; Seidah et al., supra. PCSK9 is expressed in cells capable ofproliferation and differentiation including, for example, hepatocytes,kidney mesenchymal cells, intestinal ileum, and colon epithelia as wellas embryonic brain telencephalon neurons; Seidah et al., supra.

Original synthesis of PCSK9 is in the form of an inactive enzymeprecursor, or zymogen, of ˜72-kDa which undergoes autocatalytic,intramolecular processing in the endoplasmic reticulum (“ER”) toactivate its functionality. This internal processing event has beenreported to occur at the SSVFAQ↓SIPWNL¹⁵⁸ motif (SEQ ID NOs: 19 and 20,respectively); Benjannet et al., 2004 J. Biol. Chem. 279:48865-48875.Such internal processing has been reported as a requirement of exit fromthe ER; Benjannet et al., supra; Seidah et al., supra. The cleaved and,thereby, activated protein is secreted in association with the cleavedpeptide; supra.

The sequence for human PCSK9 (˜22-kb long with 12 exons encoding a 692amino acid protein) can be found in one instance at Deposit No.NP_(—)777596.2. Human, mouse and rat PCSK9 nucleic acid sequences havebeen deposited; see, e.g., GenBank Accession Nos.: AX127530 (alsoAX207686), NP_(—)705793 (also Q80W65), and P59996, respectively. PCSK9possesses several domains found in other proprotein convertases,including an N-terminal signal sequence, a pro domain, a catalyticdomain and a cysteine-rich C terminal domain. The PCSK9 catalytic domainshares high sequence similarity with the proteinase K family ofsubtilases and, notably, a catalytic triad of D186, H226 and S386.

PCSK9 is disclosed and/or claimed in several patent publicationsincluding, but not limited to the following: PCT Publication Nos. WO01/31007, WO 01/57081, WO 02/14358, WO 01/98468, WO 02/102993, WO02/102994, WO 02/46383, WO 02/90526, WO 01/77137, and WO 01/34768; USPublication Nos. US 2004/0009553 and US 2003/0119038, and EuropeanPublication Nos. EP 1 440 981, EP 1 067 182, and EP 1 471 152.

PCSK9 has been ascribed a role in the differentiation of hepatic andneuronal cells (Seidah et al., supra.), is highly expressed in embryonicliver, and has been strongly implicated in cholesterol homeostasis.Studies have suggested a specific role for PCSK9 in cholesterolbiosynthesis or uptake. In a study of cholesterol-fed rats, Maxwell etal. found that PCSK9 was downregulated in a similar manner to threeother genes involved in cholesterol biosynthesis, Maxwell et al., 2003J. Lipid Res. 44:2109-2119. The expression of PCSK9 has, in fact, beenshown to be regulated by sterol regulatory element-binding proteins(“SREBP”), as seen with other genes involved in cholesterol metabolism;supra. Later support for these findings came about through a study ofPCSK9 transcriptional regulation which demonstrated that such regulationwas quite typical of other genes implicated in lipoprotein metabolism;Dubuc et al., 2004 Arterioscler. Thromb. Vasc. Biol. 24:1454-1459.Statins have been shown to upregulate PCSK9 expression in a mannerattributed to the cholesterol-lowering effects of the drugs; supra.Moreover, it has been shown that PCSK9 promoters possess two conservedsites involved in cholesterol regulation, a sterol regulatory elementand an Sp1 site; supra.

Several lines of evidence demonstrate that PCSK9, in particular, lowersthe amount of hepatic LDLR protein and thus compromises the liver'sability to remove LDL cholesterol from the circulation.Adenovirus-mediated overexpression of PCSK9 in the livers of miceresults in the accumulation of circulating LDL-C due to a dramatic lossof hepatic LDLR protein, with no effect on LDLR mRNA levels; Benjannetet al., 2004 J. Biol. Chem. 279:48865-48875; Maxwell & Breslow, 2004PNAS 101:7100-7105; Park et al., 2004 J. Biol. Chem. 279:50630-50638;and Lalanne et al., 2005 J. Lipid Res. 46:1312-1319. The effect of PCSK9overexpression on raising circulating LDL-C levels in mice is completelydependent on the expression of LDLR, again, indicating that theregulation of LDL-C by PCSK9 is mediated through downregulation of LDLRprotein. In agreement with these findings, mice lacking PCSK9 or inwhich PCSK9 mRNA has been lowered by antisense oligonucleotideinhibitors have higher levels of hepatic LDLR protein and a greaterability to clear circulating LDL-C; Rashid et al., 2005 PNAS102:5374-5379; and Graham et al., 2007 J. Lipid Res. 48(4):763-767. Inaddition, lowering PCSK9 levels in cultured human hepatocytes by siRNAalso results in higher LDLR protein levels and an increased ability totake up LDL-C; Benjannet et al., 2004 J. Biol. Chem. 279:48865-48875;and Lalanne et al., 2005 J. Lipid Res. 46:1312-1319. Together, thesedata indicate that PCSK9 action leads to increased LDL-C by loweringLDLR protein levels.

A number of mutations in the gene PCSK9 have also been conclusivelyassociated with autosomal dominant hypercholesterolemia (“ADH”), aninherited metabolism disorder characterized by marked elevations of lowdensity lipoprotein (“LDL”) particles in the plasma which can lead topremature cardiovascular failure; see Abifadel et al., 2003 NatureGenetics 34:154-156; Timms et al., 2004 Hum. Genet. 114:349-353; Leren,2004 Clin. Genet. 65:419-422. A later-published study on the S127Rmutation of Abifadel et al., supra, reported that patients carrying sucha mutation exhibited higher total cholesterol and apoB100 in the plasmaattributed to (1) an overproduction of apoB100-containing lipoproteins,such as low density lipoprotein (“LDL”), very low density lipoprotein(“VLDL”) and intermediate density lipoprotein (“IDL”), and (2) anassociated reduction in clearance or conversion of said lipoproteins;Ouguerram et al., 2004 Arterioscler. Thromb. Vasc. Biol. 24:1448-1453.

Accordingly, there can be no doubt that PCSK9 plays a role in theregulation of LDL. The expression or upregulation of PCSK9 is associatedwith increased plasma levels of LDL cholesterol, and the correspondinginhibition or lack of expression of PCSK9 is associated with reduced LDLcholesterol plasma levels. Decreased levels of LDL cholesterolassociated with sequence variations in PCSK9 have been found to conferprotection against coronary heart disease; Cohen, 2006 N. Engl. J. Med.354:1264-1272.

The identification of compounds and/or agents effective in the treatmentof cardiovascular affliction is highly desirable. In clinical trials,reductions in LDL cholesterol levels have been directly related to therate of coronary events; Law et al., 2003 BMJ 326:1423-1427. Morerecently, the moderate lifelong reduction in plasma LDL cholesterollevels was found to correlate with a substantial reduction in theincidence of coronary events; Cohen et al., supra. This was the caseeven in populations with a high prevalence of non-lipid-relatedcardiovascular risk factors; supra. Accordingly, there is great benefitto be reaped from the managed control of LDL cholesterol levels.

The present invention advances these interests by providing antagonistsof PCSK9 of use for inhibiting the activities of PCSK9 and thecorresponding role PCSK9 plays in various therapeutic conditions.

SUMMARY OF THE INVENTION

The present invention relates to antagonists of PCSK9 and, in particularembodiments, those antagonists that inhibit both human and murine PCSK9and those exhibiting preferential targeting of processed PCSK9. Broadly,protein-specific antagonists of PCSK9 (or “PCSK9-specific antagonists”as referred to herein) are PCSK9 protein binding molecules or moleculeseffective in the selective binding of PCSK9 and inhibition of PCSK9function. These molecules are of import in the treatment of conditionsassociated with or impacted by PCSK9 function, including, but notlimited to hypercholesterolemia, coronary heart disease, metabolicsyndrome, acute coronary syndrome and related conditions. PCSK9-specificantagonists are characterized by selective recognition and binding toPCSK9. PCSK9-specific antagonists do not show significant binding toproteins other than PCSK9, other than in those specific instances wherethe antagonist is supplemented or designed to confer an additional,distinct specificity to the PCSK9-specific binding component.

PCSK9-specific antagonists forming particular embodiments hereofcomprise (a) a heavy chain variable region comprising a CDR3 domaincomprising SEQ ID NO: 17 or an equivalent of SEQ ID NO: 17, saidequivalent characterized as having one or more conservative amino acidsubstitutions in the CDR3 domain; and/or (b) a light chain variableregion comprising a CDR3 domain comprising SEQ ID NO: 7 or an equivalentof SEQ ID NO: 7, said equivalent characterized as having one or moreconservative amino acid substitutions in the CDR3 domain. In specificembodiments, PCSK9-specific antagonists bind to human and/or murinePCSK9 with a K_(D) of 1.2×10⁻⁶ M or less. In more specific embodiments,PCSK9-specific antagonists bind to human and/or murine PCSK9 with aK_(D) of 1×10⁻⁷ M or less. In additional embodiments, PCSK9-specificantagonists bind to human and/or murine PCSK9 with a K_(D) of 1×10⁻⁸ Mor less. In further embodiments, PCSK9-specific antagonists bind tohuman and/or murine PCSK9 with a K_(D) of 5×10⁻⁹ M or less, or of 1×10⁻⁹M or less. In select embodiments, PCSK9-specific antagonists bind tohuman and/or murine PCSK9 with a K_(D) of 1×10⁻¹⁰ M or less, a K_(D) of1×10⁻¹¹ M or less, or a K_(D) of 1×10⁻¹² M or less. In specificembodiments, PCSK9-specific antagonists do not bind proteins other thanPCSK9 at the above levels indicated for binding to PCSK9.

Particular embodiments of the present invention include PCSK9-specificantagonists which exhibit binding to PCSK9 at one of the aboveprescribed levels and compete for binding to PCSK9 with 1B20 antibodymolecules. 1B20 antibody molecules form important PCSK9-specificantagonists hereof. 1B20 antibody molecules are characterized ascomprising a (i) heavy chain variable region (“VH”) comprising SEQ IDNO: 11; and (ii) a light chain variable region (“VL”) comprising SEQ IDNO: 27. Said VH and VL regions comprise the full complement of disclosedCDRs 1, 2 and 3 for the VH (SEQ ID NOs: 13, 15 and 17) and VL regions(SEQ ID NOs: 3, 5 and 7), respectively. Examples of 1B20 antibodymolecules include without limitation: (i) a Fab which comprises a lightchain comprising SEQ ID NO: 1 and an Fd chain comprising amino acidscomprising amino acids 1-221 of SEQ ID NO: 9 (or SEQ ID NO: 9); and (ii)a full length antibody molecule which comprises a light chain comprisingSEQ ID NO: 26 and a heavy chain comprising SEQ ID NO: 25.

PCSK9-specific antagonists are effective in counteractingPCSK9-dependent inhibition of cellular LDL-uptake, and particularlyhuman and/or murine PCSK9-dependent inhibition of cellular LDL uptake.Repeatedly, PCSK9-specific antagonist 1B20 has demonstrateddose-dependent inhibition of the effects of PCSK9 on LDL uptake.Accordingly, the disclosed PCSK9-specific antagonists are of import forlowering plasma LDL cholesterol levels. The disclosed antagonists alsohave utility for various diagnostic purposes, including the detectionand quantification of PCSK9. Select 1B20 antagonists are, in particular,useful because of their cross-reactivity with both human and murinePCSK9. This quality enables particular 1B20 antagonists to be studiedpharmacologically in murine models without having to ensure that themice express human PCSK9. In such experiments, the murine model issufficiently representative of the native activity of the targetedprotein and the antagonist's inhibition thereof.

In specific embodiments, the present invention encompassesPCSK9-specific antagonists. In particular embodiments, the presentinvention encompasses antibody molecules comprising the disclosed heavyand/or light chain variable regions, equivalents of said regions havingone or more conservative amino acid substitutions, and homologs thereof.Select embodiments comprise isolated PCSK9-specific antagonists thatcomprise disclosed CDR domains or sets of the heavy and/or light chainCDR domains, and equivalents of such domains characterized as having oneor more conservative amino acid substitutions. As will be appreciated bythose skilled in the art, fragments of PCSK9-specific antagonists thatretain the ability to antagonize PCSK9 may be inserted into variousframeworks; see, e.g., U.S. Pat. No. 6,818,418 and references containedtherein, the collective disclosures of which are incorporated herein byreference, which discuss various scaffolds which may be used to displayantibody loops previously selected on the basis of antigen binding. Inthe alternative, genes encoding for VL and VH may be joined, usingrecombinant methods, for example using a synthetic linker that enablesthem to be made as a single protein chain in which the VL and VH regionspair to form monovalent molecules, otherwise known as single chain Fvs(“ScFVs”); see, e.g., Bird et al., 1988 Science 242: 423-426, and Hustonet al., 1988 Proc. Natl. Acad. Sci. USA 85:5879-5883, the disclosures ofwhich are incorporated herein by reference.

PCSK-9 specific antagonists and fragments may be in the form of variousnon-antibody-based scaffolds, including but not limited to avimers(Avidia); DARPins (Molecular Partners); Adnectins (Adnexus), Anticalins(Pieris) and Affibodies (Affibody). The use of alternative scaffolds forprotein binding is well appreciated in the scientific literature, see,e.g., Binz & Plückthun, 2005 Curr. Opin. Biotech. 16:1-11; thedisclosure of which is incorporated herein by reference. Accordingly,non-antibody-based scaffolds or antagonist molecules comprising (i) thedisclosed heavy and/or light chain variable region CDR3 sequences (SEQID NOs: 17 and 7, respectively), (ii) the disclosed heavy chain variableCDR1, CDR2 and CDR3 sequences or the disclosed light chain variableCDR1, CDR2 and CDR3 sequences: CDR1 (SEQ ID NOs: 13 and 3,respectively), CDR2 (SEQ ID NOs: 15 and 5, respectively) and CDR3 (SEQID NOs; 17 and 7, respectively, (iii) the full complement (SEQ ID NOs;13, 15, 17, 3, 5 and 7) of disclosed heavy and light chain CDRs within avariable region framework of a human heavy and/or light chain sequence,respectively, or (iv) the disclosed heavy and/or light chain variableregions SEQ ID NO: 11 and/or SEQ ID NO: 27 form important embodiments ofthe present invention, where such scaffolds or antagonist moleculesexhibit selectivity for PCSK9 and counteract PCSK9-dependent inhibitionof cellular LDL-uptake. In another aspect, the present inventionprovides nucleic acid encoding the disclosed PCSK9-specific antagonistsand, in particular embodiments, PCSK9-specific antagonists whichcomprise the disclosed heavy and light chains, the disclosed variableheavy and light regions and select components thereof (including CDRs 1,2 and/or 3), particularly the disclosed respective CDR3 regions. Inanother aspect, the present invention provides vectors comprising saidnucleic acid. The present invention, additionally, provides isolatedcell(s) comprising nucleic acid encoding disclosed PCSK9-specificantagonists. In another aspect, the present invention provides isolatedcell(s) comprising a polypeptide or vector of the present invention.

The present invention provides methods for making PCSK9-specificantagonists disclosed herein including but not limited to antibodies,antigen binding fragments, derivatives, chimeric molecules, fusions ofany of the foregoing with another polypeptide, or alternativestructures/compositions capable of specifically binding PCSK9 whichcomprise the disclosed sequences. The methods comprise: (i) incubating acell comprising nucleic acid encoding the PCSK9-specific antagonist(s),or which comprises individual nucleic acids encoding one or morecomponents thereof, said nucleic acids which, when expressed,collectively produce the antagonist(s), under conditions that allow forthe expression and/or assembly of the PCSK9-specific antagonist(s), and(ii) isolating said antagonist(s) from the cell. One of skill in the artcan obtain PCSK9-specific antagonists disclosed herein using standardrecombinant DNA techniques as well.

The present invention provides a method for antagonizing the activity orfunction of PCSK9 or a noted effect of PCSK9 which comprises contactinga cell, population of cells, or tissue sample of interest expressingPCSK9 (or treated with or having therein human or murine PCSK9) with aPCSK9-specific antagonist disclosed herein under conditions that allowsaid antagonist to bind to PCSK9. Specific embodiments of the presentinvention include such methods wherein the cell is a human or murinecell. Additional embodiments are wherein the cell expresses human ormurine-derived PCSK9.

In another aspect, the present invention provides a method forantagonizing the activity or function of PCSK9 or a noted effect ofPCSK9 in a subject exhibiting a condition associated with PCSK9activity, or a condition where the functioning of PCSK9 iscontraindicated for a particular subject, which comprises administeringto the subject a therapeutically effective amount of a PCSK9-specificantagonist of the present invention in a pharmaceutical or othercomposition.

The present invention, thus, encompasses a method of treating acondition associated with PCSK9 activity, or a condition wherein thefunctioning of PCSK9 is contraindicated for a particular subject, whichcomprises administering to the subject a therapeutically effectiveamount of a PCSK9-specific antagonist of the present invention in apharmaceutical or other composition. In select embodiments, thecondition is hypercholesterolemia, coronary heart disease, metabolicsyndrome, acute coronary syndrome or related conditions.

In specific embodiments, the present invention encompasses a method ofadministering a disclosed PCSK9-specific antagonist to a subject whichcomprises delivering a therapeutically effective amount of apharmaceutical or other composition comprising a PCSK9-specificantagonist as disclosed herein.

In another aspect, the present invention provides a pharmaceuticalcomposition or other composition comprising a PCSK9-specific antagonistof the invention characterized as comprising a pharmaceuticallyacceptable carrier including but not limited to an excipient, diluent,stabilizer, buffer, or alternative designed to facilitate administrationof the antagonist in the desired amount to the treated individual.

The following table offers a generalized outline of the sequencesdiscussed in the present application. The Sequence Listing including allnotations, sequences and features forms an express part of thedisclosure hereof:

TABLE 1 SEQ ID NO: DESCRIPTION SEQ ID NO: 1 LIGHT CHAIN (“LC”); 1B20 SEQID NO: 2 LIGHT CHAIN (“LC”) NUCLEIC ACID; 1B20 SEQ ID NO: 3 VL CDR1;1B20 SEQ ID NO: 4 VL CDR1 NUCLEIC ACID; 1B20 SEQ ID NO: 5 VL CDR2; 1B20SEQ ID NO: 6 VL CDR2 NUCLEIC ACID; 1B20 SEQ ID NO: 7 VL CDR3; 1B20 SEQID NO: 8 VL CDR3 NUCLEIC ACID; 1B20 SEQ ID NO: 9 Fd CHAIN inclusive oflinkers and tags; 1B20 SEQ ID NO: 10 Fd CHAIN NUCLEIC ACID; 1B20 SEQ IDNO: 11 VH; 1B20 SEQ ID NO: 12 VH NUCLEIC ACID; 1B20 SEQ ID NO: 13 VHCDR1; 1B20 SEQ ID NO: 14 VH CDR1 NUCLEIC ACID; 1B20 SEQ ID NO: 15 VHCDR2; 1B20 SEQ ID NO: 16 VH CDR2 NUCLEIC ACID; 1B20 SEQ ID NO: 17 VHCDR3; 1B20 SEQ ID NO: 18 VH CDR3 NUCLEIC ACID; 1B20 SEQ ID NO: 19FRAGMENT OF PROCESSING SITE SEQ ID NO: 20 FRAGMENT OF PROCESSING SITESEQ ID NO: 21 Constant domain of IgG1 SEQ ID NO: 22 Constant domain ofIgG2 SEQ ID NO: 23 Constant domain of IgG4 SEQ ID NO: 24 Constant domainof IgG2m4 SEQ ID NO: 25 1B20 IgG2m4 Heavy Chain (“HC”) SEQ ID NO: 261B20 IgG Light (Kappa) Chain SEQ ID NO: 27 VL; 1B20 SEQ ID NO: 28 VLNUCLEIC ACID; 1B20 SEQ ID NO: 29 1B20 IgG2m4 HC NUCLEIC ACID SEQ ID NO:30 1B20 IgG LC NUCLEIC ACID SEQ ID NO: 31 PRIMER SEQ ID NO: 32 PRIMERSEQ ID NO: 33 PRIMER SEQ ID NO: 34 PRIMER SEQ ID NO: 35 1B20 IgG2m4 HCPLASMID SEQ ID NO: 36 1B20 IgG LC PLASMID SEQ ID NO: 37 1B20 Variant VHCDR1 Sequence SEQ ID NO: 38 1B20 Variant VH CDR2 Sequence SEQ ID NO: 391B20 Variant VH CDR3 Sequence SEQ ID NO: 40 1B20 Variant VL CDR1Sequence SEQ ID NO: 41 1B20 Variant VL CDR2 Sequence SEQ ID NO: 42 1B20Variant VL CDR3 Sequence SEQ ID NO: 43 VL; 1B20 Variant Sequence SEQ IDNO: 44 VH; 1B20 Variant Sequence SEQ ID NO: 45 VH; 1B20 Variant SequenceF120 SEQ ID NO: 46 VH; 1B20 Variant Sequence F116 SEQ ID NO: 47 VH; 1B20Variant Sequence F119 SEQ ID NO: 48 VH; 1B20 Variant Sequence F113 SEQID NO: 49 VH; 1B20 Variant Sequence E2 SEQ ID NO: 50 VH; 1B20 VariantSequence G4 SEQ ID NO: 51 VH; 1B20 Variant Sequence F4 SEQ ID NO: 52 VH;1B20 Variant Sequence B9 SEQ ID NO: 53 VH; 1B20 Variant Sequence C3 SEQID NO: 54 VH; 1B20 Variant Sequence F2 SEQ ID NO: 55 VH; 1B20 VariantSequence F7 SEQ ID NO: 56 VH; 1B20 Variant Sequence A7 SEQ ID NO: 57 VH;1B20 Variant Sequence G8 SEQ ID NO: 58 VH; 1B20 Variant Sequence H4 SEQID NO: 59 VH; 1B20 Variant Sequence D5 SEQ ID NO: 60 VH; 1B20 VariantSequence D4 SEQ ID NO: 61 VH; 1B20 Variant Sequence B4 SEQ ID NO: 62 VH;1B20 Variant Sequence H1 SEQ ID NO: 63 VH; 1B20 Variant Sequence G2 SEQID NO: 64 VH; 1B20 Variant Sequence A1 SEQ ID NO: 65 VH; 1B20 VariantSequence A4 SEQ ID NO: 66 VH; 1B20 Variant Sequence C2 SEQ ID NO: 67 VH;1B20 Variant Sequence H5 SEQ ID NO: 68 VH; 1B20 Variant Sequence F6 SEQID NO: 69 VH; 1B20 Variant Sequence B6 SEQ ID NO: 70 VH; 1B20 VariantSequence B1 SEQ ID NO: 71 VH; 1B20 Variant Sequence F1 SEQ ID NO: 72 VH;1B20 Variant Sequence A8 SEQ ID NO: 73 VH; 1B20 Variant Sequence B3 SEQID NO: 74 VH; 1B20 Variant Sequence F8 SEQ ID NO: 75 VH; 1B20 VariantSequence H8 SEQ ID NO: 76 VH; 1B20 Variant Sequence B5 SEQ ID NO: 77 VH;1B20 Variant Sequence E1 SEQ ID NO: 78 VH; 1B20 Variant Sequence E8 SEQID NO: 79 VH; 1B20 Variant Sequence C1 SEQ ID NO: 80 VH; 1B20 VariantSequence H3 SEQ ID NO: 81 VH; 1B20 Variant Sequence A9 SEQ ID NO: 82 VH;1B20 Variant Sequence G7 SEQ ID NO: 83 VH; 1B20 Variant Sequence C6 SEQID NO: 84 VH; 1B20 Variant Sequence G6 SEQ ID NO: 85 VH; 1B20 VariantSequence E4 SEQ ID NO: 86 VH; 1B20 Variant Sequence E5 SEQ ID NO: 87 VH;1B20 Variant Sequence C7 SEQ ID NO: 88 VH; 1B20 Variant Sequence E3 SEQID NO: 89 VH; 1B20 Variant Sequence D3 SEQ ID NO: 90 VH; 1B20 VariantSequence D8 SEQ ID NO: 91 VH; 1B20 Variant Sequence C8 SEQ ID NO: 92 VH;1B20 Variant Sequence E5 SEQ ID NO: 93 VH; 1B20 Variant Sequence B8 SEQID NO: 94 VH; 1B20 Variant Sequence H7 SEQ ID NO: 95 VH; 1B20 VariantSequence A5 SEQ ID NO: 96 VH; 1B20 Variant Sequence A3 SEQ ID NO: 971B20 VARIANT VH CDR2 SEQUENCE SEQ ID NO: 98 1B20 VARIANT VH CDR3SEQUENCE SEQ ID NO: 99 1B20 VARIANT VL CDR1 SEQUENCE SEQ ID NO: 100 1B20VARIANT VL CDR2 SEQUENCE SEQ ID NO: 101 1B20 VARIANT VL CDR3 SEQUENCESEQ ID NO: 102 VH; 1B20 Variant Sequence N59K SEQ ID NO: 103 VH; 1B20Variant Sequence N59Q SEQ ID NO: 104 VH; 1B20 Variant Sequence N59R SEQID NO: 105 VH; 1B20 Variant Sequence W101A SEQ ID NO: 106 VH; 1B20Variant Sequence W101F SEQ ID NO: 107 VH; 1B20 Variant Sequence W101YSEQ ID NO: 108 VL; 1B20 Variant Sequence SEQ ID NO: 109 VH; 1B20 VariantSequence

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates Fab expression vector pMORPH_x9_MH encoding the6CX1B20 (“1B20”) Fab heavy and light chains.

FIG. 2 illustrates the activity of 1B20 in a PCSK9-LDLR interactionTR-FRET assay. Both the Fab and IgG of 1B20 are potent and inhibit theinteraction fully. For the experiment, [AF647-PCSK9]=10 nM, [Eu-sLDLR]˜4nM (˜20000 counts at FI₆₂₀ nm).

FIGS. 3A-3D illustrate (i) 1B20 (Fab)'s dose-dependent inhibition ofmurine PCSK9-dependent loss of cellular LDL-uptake (FIG. 3A); (ii) 1B20(Fab)'s dose-dependent inhibition of human PCSK9-dependent loss ofcellular LDL-uptake (FIG. 3B); (iii) 1B20 (IgG)'s dose-dependentinhibition of murine PCSK9-dependent loss of cellular LDL-uptake (FIG.3C); and (iv) 1B20 (IgG)'s dose-dependent inhibition of humanPSCK9-dependent loss of cellular LDL-uptake (FIG. 3D). 1B20 clearlycross-reacts with both human and mouse PCSK9. FIGS. 3A-3D have twocontrols: (i) a cell only control, showing the basal level of cellularLDL uptake, and (ii) a PCSK9 (5 μg/ml) control which shows the level ofPCSK9-dependent loss of LDL-uptake. The titration experiments whichcontain 1B20 and PCSK9 were done at a fixed concentration of PCSK9 (5μg/ml) and increasing concentrations of 1B20 shown in the graphs. Asshown, 1B20 can inhibit the effect of PCSK9 on cellular LDL uptake.IC₅₀s for 1B20 (Fab) are 152 nM (n=5) and 145 nM (n=5) for mouse andhuman PCSK9 protein, respectively. IC₅₀s for 1B20 (IgG) are 13 nM and 22nM for mouse and human PCSK9 protein, respectively.

FIG. 4 illustrates inhibition of PCSK9 internalization by 1B20 (IgG).HEK293 cells were plated and AlexaFluor-labeled PCSK9 and LDL were thenadded to cells and incubated at 37° C. for 4 hrs. Following incubation,the amount of PCSK9 or LDL internalized by cells was determined usingcofocal microscopy. Controls included the addition of cells alone (Notreatment), and only AF-labeled PCSK9 in addition to 50× (250 μg/ml)unlabeled PCSK9 (50× Cold Wt). In addition to 5 μg/ml wild-typeAF-labeled PCSK9 and 10 μg/ml AF-labeled LDL, increasing amounts of the1B20 IgG was added, resulting in subsequent inhibition of PCSK9internalization into cells. Together, these studies demonstrate that the1B20 IgG prevents PCSK9 internalization into cells.

FIG. 5 illustrates the LDL levels for each mouse represented by a set ofconnected symbols; the change in LDL (postbleed—prebleed) being shown asan average for each treatment group (Δ mg/dL). Treatment with PBS had noeffect on LDL measurements (−4 mg/dL, 5% reduction). In contrast, serumLDL was reduced 20% with 1B20 whole IgG (−19 mg/dL).

FIG. 6 illustrates a sequence comparison of the constant domains of IgG1(SEQ ID NO: 21; Fc domain of which is represented by residues 110-130 ofSEQ ID NO: 21), IgG2 (SEQ ID NO: 22, Fc domain of which is representedby residues 107-326 of SEQ ID NO: 22), IgG4 (SEQ ID NO: 23; Fc domain ofwhich is represented by residues 107-327 of SEQ ID NO: 23) and IgG2m4(SEQ ID NO: 24; Fc domain of which is represented by residues 107-326 ofSEQ ID NO: 24) isotypes.

FIG. 7 illustrates 1B20 lowers LDL-C by ˜50% in rhesus at 1, 3 and 10mpk. Plotted are % LDL changes in serum at the different time pointstested, post a single IV dose of antibody treatment.

FIG. 8 illustrates the pharmacokinetic profile of 1B20 at the doselevels shown. Plotted is the serum drug (1B20) levels at time pointstested following a single IV dose of antibody. The half-life of 1B20 is39 hr.

FIG. 9 illustrates the change in total aggregates at 37° C. from time 0as measured by SEC.

FIG. 10 illustrates Dynamic Light Scattering (“DLS”) measurements ofMK-2370 in various formulations after one, two and three months ofstorage at 37° C.

FIG. 11 illustrates the change in total aggregation at 25° C. from time0 in liquid with a protein concentration of 50 mg/mL as measured bySEC-HPLC.

FIG. 12 illustrates the change in total aggregation at 25° C. from time0 in liquid with a protein concentration of 50 mg/mL as measured bySEC-HPLC.

FIG. 13 illustrates total aggregation observed at 50, 100 and 150 mg/mLafter 6 months storage at 25° C. in the liquid form.

FIG. 14 illustrates the change in total aggregation at 25° C. from time0 at 50 mg/mL as measured by SEC-HPLC; *6/100/100 data from a differentstudy.

FIG. 15 illustrates total aggregates of various concentrations of mAb3at 25° C. as measured by SEC-HPLC.

FIG. 16 illustrates total aggregation observed at 25° C. with a proteinconcentration of 50 mg/mL as measured by SEC-HPLC.

FIG. 17 illustrates total aggregation observed at 25° C. with a proteinconcentration of 50 mg/mL as measured by SEC-HPLC.

FIG. 18 illustrates the change in total aggregation at varioustemperature over three months of storage at 50 mg/mL in lyophilized formas measured by SEC-HPLC.

FIG. 19 illustrates total aggregation at 50 mg/mL in lyophilized form asmeasured by SEC-HPLC.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to antagonists of PCSK9 and, in particularembodiments, those antagonists that inhibit both human and murine PCSK9and those that preferentially target processed PCSK9. Protein-specificantagonists of PCSK9 (or “PCSK9-specific antagonists”) in accordanceherewith are effective in the selective binding to and inhibition ofPCSK9 function and, thus, are of import in the treatment of conditionsassociated with or impacted by PCSK9 function, including, but notlimited to, hypercholesterolemia, coronary heart disease, metabolicsyndrome, acute coronary syndrome and related conditions. Use of theterm “antagonist” refers to the fact that the subject molecule canantagonize the functioning of PCSK9. Use of the term “antagonizing” orderivatives thereof refers to the act of opposing, counteracting,inhibiting, neutralizing or curtailing one or more functions of PCSK9.Reference herein to PCSK9 function or PCSK9 activity refers to anyfunction or activity that is driven by, requires, or is exacerbated orenhanced by PCSK9. PCSK9-specific antagonists as described herein haveproven to be effective for counteracting human and/or murinePCSK9-dependent inhibition of cellular LDL-uptake.

One important embodiment hereof relates to 1B20 antibody molecules. Such1B20 antibody molecules are characterized as comprising a (i) heavychain variable region (“VH”) comprising SEQ ID NO: 11; and (ii) a lightchain variable region (“VL”) comprising SEQ ID NO: 27. Said VH and VLregions comprise the full complement of disclosed CDRs 1, 2 and 3 forthe VH (SEQ ID NOs: 13, 15 and 17) and VL regions (SEQ ID NOs: 3, 5 and7), respectively. Examples of 1B20 antibody molecules include withoutlimitation: (i) a Fab which comprises a light chain comprising SEQ IDNO: 1 and an Fd chain comprising amino acids 1-221 of SEQ ID NO: 9 (orSEQ ID NO: 9); and (ii) a full length antibody molecule which comprisesa light chain comprising SEQ ID NO: 26 and a heavy chain comprising SEQID NO: 25. The select group of 1B20 antibodies demonstrate thatPCSK9-specific antagonists as disclosed herein effectively inhibit bothhuman and murine PCSK9 and may be studied pharmacologically in murinemodels absent the expression of human PCSK9.

The CDR definitions arrived at and disclosed herein were defined usingthe Morphosys software program Sequence Analysis Software (“SAS”).Applicants wish to note, however, that various other methods areavailable to delineate and define the start and end points of the CDRsequences, including but not limited to Kabat, 1991 Sequences ofProteins of Immunological Interest, 5^(th) edit., NIH Publication no.91-3242 U.S. Department of Health and Human Services; Clothia et al.,1987 J. Mol. Biol. 196:901-917; Clothia et al., 1989 Nature 342:877-883;Lefranc, 1997 Immunol. Today, 18:509; and Chen et al., 1999 J. Mol.Biol. 293:865-881. These and other methods have been reviewed and arewell within the realm of skills possessed by those in the art; see,e.g., Honegger & Plückthun, 2001 J. Mol. Biol. 309:657-670. While thecurrent inventors have employed the SAS software to define the CDRs, thepresent invention fully encompasses the different definitions around thesequences and the varying CDR delineations arrived at through use of anydifferent analysis software or methods. Said use and resulting CDRdefinitions based on the presently disclosed sequences is fully withinthe scope of the present disclosure and anticipated herein.

PCSK9-specific molecules also have utility for various diagnosticpurposes in the detection and quantification of PCSK9.

Disclosed PCSK9-specific antagonists are, furthermore, unique in thatselect embodiments have demonstrated a preferential recognition ofprocessed PCSK9, the active form of PCSK9.

PCSK9-specific antagonists as disclosed herein are desirable moleculesfor lowering plasma LDL cholesterol levels and are of utility for anyprimate, mammal or vertebrate of commercial or domestic veterinaryimportance. PCSK9-specific antagonists are of utility as well to inhibitthe activity of PCSK9 in any population of cells or tissues possessingthe LDL receptor. The utility of the disclosed antagonists is directlymeasurable by assays readily available to the skilled artisan. Means formeasuring LDL uptake are described in the literature; see, e.g., Barak &Webb, 1981 J. Cell Biol. 90:595-604, and Stephan & Yurachek, 1993 J.Lipid Res. 34:325330. In addition, means for measuring LDL cholesterolin plasma is well described in the literature; see, e.g., McNamara etal., 2006 Clinica Chimica Acta 369:158-167. The particular impact of thedisclosed antagonists on cellular LDL uptake may also be measuredthrough a method which comprises providing purified PCSK9 and labeledLDL particles to a cell sample; providing a PCSK9 antagonist to the cellsample; incubating said cell sample for a period of time sufficient toallow LDL particle uptake by the cells; quantifying the amount of labelincorporated into the cell; and identifying those antagonists thatresult in an increase in the amount of quantified label taken up by thecells as compared with that observed when PCSK9 is administered alone.An additional method for measuring the impact of the disclosedantagonists comprises providing purified PCSK9 and labeled LDL particlesto a cell sample; providing a PCSK9 antagonist to the cell sample;incubating said cell sample for a period of time sufficient to allow LDLparticle uptake by the cells; isolating cells of the cell sample byremoving the supernate; reducing non-specific association of labeled LDLparticles (whether to the plate, the cells, or anything other than theLDL receptor); lysing the cells; quantifying the amount of labelretained within the cell lysate; and identifying those antagonists thatresult in an increase in the amount of quantified label taken up by thecells as compared with that observed when PCSK9 is administered alone.Antagonists that result in an increase in the amount of quantified labelare PCSK9 antagonists.

Any type of cell bearing the LDL receptor can be employed in the abovemethods including, but not limited to HEK cells, HepG2 cells, and CHOcells. LDL particles derived from any source are of use in theabove-described assays. In particular assays, the LDL particles arefresh particles derived from blood. This can be accomplished by anymethod available to the skilled artisan including, but not limited to,the method of Havel et al., 1955 J. Clin. Invest. 34: 1345-1353. The LDLparticles may be labeled with fluorescence. The labeled LDL particlesmay have incorporated therein visible wavelength excited fluorophore3,3′-dioctadecylindocarbocyanine iodide (dil(3)) to form the highlyfluorescent LDL derivative dil(3)-LDL. Any label which enables theskilled artisan to detect LDL in the cellular lysate may be used. An LDLanalog may be used that would only become detectable (e.g., becomefluorescent or fluoresce at a different wavelength, etc.) whenmetabolized intracellularly or, for instance, if it were to becomeassociated with (or dissociated from) other molecules in the process ofbecoming internalized (e.g. a FRET assay, in which an LDL analog wouldbecome associated with a secondary fluor, or else be dissociated from aquencher). Any means available in the art for detecting internalizationof labeled LDL particles can be employed. The incubation time for theLDL particles and PCSK9 with the cells is an amount of time sufficientto allow LDL particle uptake by the cells. This time may be within therange of 5 minutes to 360 minutes. The concentration of PCSK9 added tothe cells may be in the range of 1 nM to 5 μM and, in specific methods,be in the range of 0.1 nM to 3 μM. One specific means by which theskilled artisan can determine a range of concentrations for a particularPCSK9 protein is to develop a dose response curve in the LDL-uptakeassay. A concentration of PCSK9 can be selected that promotes close tomaximal loss of LDL-uptake and is still in the linear range of the doseresponse curve. Typically, this concentration is ˜5 times the EC-50 ofthe protein extracted from the dose response curve. The concentrationscan vary by protein.

Broadly, PCSK9-specific antagonists as defined herein selectivelyrecognize and specifically bind to PCSK9. An antibody is typically saidto specifically bind an antigen when the dissociation constant is ≦1 μM,preferably ≦100 nM and most preferably ≦10 nM. Use of the terms“selective” or “specific” herein, further, refers to the fact that thedisclosed antagonists do not show significant binding to proteins otherthan PSCK9, except in those specific instances where the antagonist issupplemented or designed to confer an additional, distinct specificityto the PCSK9-specific binding portion (as, for example, in bispecific orbifunctional molecules where the molecule is designed to bind twomolecules or effect two functions, at least one of which is tospecifically bind PCSK9). In specific embodiments, PCSK9-specificantagonists bind to human and/or murine PCSK9 with a K_(D) of 1.2×10⁻⁶ Mor less. In more specific embodiments, PCSK9-specific antagonists bindto human and/or murine PCSK9 with a K_(D) of 5×10⁻⁷ M or less, of 2×10⁻⁷M or less, or of 1×10⁻⁷ M or less. In additional embodiments,PCSK9-specific antagonists bind to human and/or murine PCSK9 with aK_(D) of 1×10⁻⁸ M or less. In further embodiments, PCSK9-specificantagonists bind to human and/or murine PCSK9 with a K_(D) of 5×10⁻⁹ Mor less, or of 1×10⁻⁹ M or less. In select embodiments, PCSK9-specificantagonists bind to human and/or murine PCSK9 with a K_(D) of 1×10⁻¹⁰ Mor less, a K_(D) of 1×10⁻¹¹ M or less, or a K_(D) of 1×10⁻¹² M or less.In specific embodiments, PCSK9-specific antagonists do not bind proteinsother than PCSK9 at the above K_(D)s. K_(D) refers to the dissociationconstant obtained from the ratio of K_(d) (the dissociation rate of aparticular binding molecule-target protein interaction) to K_(a) (theassociation rate of the particular binding molecule-target proteininteraction), or K_(d)/K_(a) which is expressed as a molar concentration(M). K_(D) values can be determined using methods well established inthe art. A preferred method for determining the K_(D) of a bindingmolecule is by using surface plasmon resonance, for example employing abiosensor system such as a Biacore™ (GE Healthcare Life Sciences)system.

PCSK9-specific antagonists disclosed herein have been shown todose-dependently inhibit human and/or murine PCSK9 dependent effects onLDL uptake. Accordingly, PCSK9-specific antagonists as disclosed hereinare characterized by their ability to counteract PCSK9-dependentinhibition of LDL uptake into cells. This uptake of LDL into cells bythe LDL receptor is referred to herein as “cellular LDL uptake”. Inspecific embodiments, PCSK9-specific antagonists counteract orantagonize human and/or murine PCSK9-dependent inhibition of LDL uptakeinto cells, exhibiting an IC₅₀ of less than 1.0×10⁻⁶ M, or, in order ofpreference, less than 1×10⁻⁷ M, 1×10⁻⁸ M, 1×10⁻⁹ M, 1×10⁻¹⁰ M, 1×10⁻¹¹ Mand 1×10⁻¹² M. The extent of inhibition by any PCSK9-specific antagonistmay be measured quantitatively in statistical comparison to a control,or via any alternative method available in the art for assessing anegative effect on, or inhibition of, PCSK9 function (i.e., any methodcapable of assessing antagonism of PCSK9 function). In specificembodiments, the inhibition is at least about 10% inhibition. In otherembodiments, the inhibition is at least 20%, 30%, 40%, 50%, 60%, 70,%,80%, 90%, or 95%. Accordingly, PCSK9-specific antagonists capable ofeffecting these levels of inhibition of PCSK9 function form particularembodiments hereof.

A PCSK9-specific antagonist in accordance herewith can be any bindingmolecule that specifically binds human and/or murine PCSK9 proteinincluding, but not limited to, antibody molecules as defined below, anyPCSK9-specific binding structure, any polypeptide or nucleic acidstructure that specifically binds PCSK9, and any of the foregoingincorporated into various protein scaffolds; including but not limitedto, various non-antibody-based scaffolds, and various structures capableof affording or allowing for selective binding to PCSK9 including butnot limited to small modular immunopharmaceuticals (or “SMIPs”; see,Haan & Maggos, 2004 Biocentury January 26); Immunity proteins (see,e.g., Chak et al., 1996 Proc. Natl. Acad. Sci. USA 93:6437-6442);cytochrome b562 (see Ku and Schultz, 1995 Proc. Natl. Acad. Sci. USA92:6552-6556); the peptide α2p8 (see Barthe et al., 2000 Protein Sci.9:942-955); avimers (Avidia; see Silverman et al., 2005 Nat. Biotechnol.23:1556-1561); DARPins (Molecular Partners; see Binz et al., 2003 J.Mol. Biol. 332:489-503; and Forrer et al., 2003 FEBS Lett. 539:2-6);Tetranectins (see, Kastrup et al., 1998 Acta. Crystallogr. D. Biol.Crystallogr. 54:757-766); Adnectins (Adnexus; see, Xu et al., 2002 Chem.Biol. 9:933-942), Anticalins (Pieris; see Vogt & Skerra, 2004Chemobiochem. 5:191-199; Beste et al., 1999 Proc. Natl. Acad. Sci. USA96:1898-1903; Lamla & Erdmann, 2003 J. Mol. Biol. 329:381-388; and Lamla& Erdmann, 2004 Protein Expr. Purif 33:39-47); A-domain proteins (seeNorth & Blacklow, 1999 Biochemistry 38:3926-3935), Lipocalins (seeSchlehuber & Skerra, 2005 Drug Discov. Today 10:23-33); Repeat-motifproteins such as Ankyrin repeat proteins (see Sedgwick & Smerdon, 1999Trends Biochem. Sci. 24:311-316; Mosavi et al., 2002 Proc. Natl. Acad.Sci. USA 99:16029-16034; and Binz et al., 2004 Nat. Biotechnol.22:575-582); Insect Defensin A (see Zhao et al., 2004 Peptides25:629-635); Kunitz domains (see Roberts et al., 1992 Proc. Natl. Acad.Sci. USA 89:2429-2433; Roberts et al., 1992 Gene 121:9-15; Dennis &Lazarus, 1994 J. Biol. Chem. 269:22129-22136; and Dennis & Lazarus, 1994J. Biol. Chem. 269:22137-22144); PDZ-Domains (see Schneider et al., 1999Nat. Biotechnol. 17:170-175); Scorpion toxins such as Charybdotoxin (seeVita et al., 1998 Biopolymers 47:93-100); 10^(th) fibronectin type IIIdomain (or 10Fn3; see Koide et al., 1998 J. Mol. Biol. 284:1141-1151,and Xu et al., 2002 Chem. Biol. 9:933-942); CTLA-4 (extracellulardomain; see Nuttall et al., 1999 Proteins 36:217-227; and Irving et al.,2001 J. Immunol. Methods 248:31-45); Knottins (see Souriau et al., 2005Biochemistry 44:7143-7155 and Lehtio et al., 2000 Proteins 41:316-322);Neocarzinostatin (see Heyd et al. 2003 Biochemistry 42:5674-5683);carbohydrate binding module 4-2 (CBM4-2; see Cicortas et al., 2004Protein Eng Des. Sel. 17:213-221); Tendamistat (see McConnell & Hoess,1995 J. Mol. Biol. 250:460-470, and Li et al., 2003 Protein Eng.16:65-72); T cell receptor (see Holler et al., 2000 Proc. Natl. Acad.Sci. USA 97:5387-5392; Shusta et al., 2000 Nat. Biotechnol. 18:754-759;and Li et al., 2005 Nat. Biotechnol. 23:349-354); Affibodies (Affibody;see Nord et al., 1995 Protein Eng. 8:601-608; Nord et al., 1997 Nat.Biotechnol. 15:772-777; Gunneriusson et al., 1999 Protein Eng.12:873-878); and other selective binding proteins or scaffoldsrecognized in the literature; see, e.g., Binz & Plückthun, 2005 Curr.Opin. Biotech. 16:1-11; Gill & Damle, 2006 Curr. Opin. Biotechnol.17:1-6; Hosse et al., 2006 Protein Science 15:14-27; Binz et al., 2005Nat. Biotechnol. 23:1257-1268; Hey et al., 2005 Trends in Biotechnol.23:514-522; Binz & Plückthun, 2005 Curr. Opin. Biotech. 16:459-469;Nygren & Skerra, 2004 J. Immunolog. Methods 290:3-28; Nygren & Uhlen,1997 Curr. Opin. Struct. Biol. 7:463-469; the disclosures of which areincorporated herein by reference. Antibodies and the use ofantigen-binding fragments is well defined and understood in theliterature. The use of alternative scaffolds for protein binding is wellappreciated in the scientific literature as well, see, e.g., Binz &Plückthun, 2005 Curr. Opin. Biotech. 16: 1-11; Gill & Damle, 2006 Curr.Opin. Biotechnol. 17:1-6; Hosse et al., 2006 Protein Science 15:14-27;Binz et al., 2005 Nat. Biotechnol. 23:1257-1268; Hey et al., 2005 Trendsin Biotechnol. 23:514-522; Binz & Plückthun, 2005 Curr. Opin. Biotech.16:459-469; Nygren & Skerra, 2004 J. Immunolog. Methods 290:3-28; Nygren& Uhlen, 1997 Curr. Opin. Struct. Biol. 7:463-469; the disclosures ofwhich are incorporated herein by reference. Accordingly,non-antibody-based scaffolds or antagonist molecules in accordanceherewith exhibiting selectivity for PCSK9 that counteractPCSK9-dependent inhibition of cellular LDL-uptake form importantembodiments of the present invention. Aptamers (nucleic acid or peptidemolecules capable of selectively binding a target molecule) are onespecific example. They can be selected from random sequence pools oridentified from natural sources such as riboswitches. Peptide aptamers,nucleic acid aptamers (e.g., structured nucleic acid, including both DNAand RNA-based structures) and nucleic acid decoys can be effective forselectively binding and inhibiting proteins of interest; see, e.g.,Hoppe-Seyler & Butz, 2000 J. Mol. Med. 78:426-430; Bock et al., 1992Nature 355:564-566; Bunka & Stockley, 2006 Nat. Rev. Microbiol.4:588-596; Martell et al., 2002 Molec. Ther. 6:30-34; Jayasena, 1999Clin. Chem. 45:1628-1650; the disclosures of which are incorporatedherein by reference.

Given 1B20's significant neutralizing activity, it is clearly ofinterest to identify other PCSK9-specific antagonists that bind to PCSK9in the same manner as 1B20. One means of identifying antagonists andparticularly antibodies that bind to the same region or epitope as 1B20or an overlapping epitope is through a competition or similar assaywhere the candidate antibody or binding molecule would have toout-compete 1B20 for the epitope. Competitive antagonists encompassedherein are molecules that inhibit (i.e., prevent or interfere with 1B20binding in comparison to a control) or reduce 1B20 binding by at least50%, 60%, 70%, and 80% in order of increasing preference (even morepreferably, at least 90% and, most preferably, at least 95%) at 1 μM orless with 1B20 at or below its K_(D), and in particular those moleculesthat antagonize (i) PCSK9 binding to the LDL receptor, (ii) PCSK9internalization into cells, or (iii) both PCSK9 binding to the LDLreceptor and PCSK9 internalization into cells. Competition betweenbinding members may be readily assayed in vitro for example using ELISAand/or by monitoring the interaction of the antibodies with PCSK9 insolution. The exact means for conducting the analysis is not critical.PCSK9 may be immobilized to a 96-well plate or may be placed in ahomogenous solution. In specific embodiments, the ability of unlabeledcandidate antibody(ies) to block the binding of labeled 1B20 can bemeasured using radioactive, enzyme or other labels. In the reverseassay, the ability of unlabeled antibodies to interfere with theinteraction of labeled 1B20 with PCSK9 wherein said 1B20 and PCSK9 arealready bound is determined. In specific embodiments, (i) PCSK9 iscontacted with labeled 1B20 (an antibody molecule which comprises a VLcomprising SEQ ID NO: 27 and a VH comprising SEQ ID NO: 11); (ii) PCSK9is contacted with the candidate antibody or pool of antibodies; and(iii) antibodies capable of interrupting or preventing complexes betweenPCSK9 and 1B20 are identified. The readout in such an example is throughmeasurement of bound label. 1B20 and the candidate antibody(ies) may beadded in any order or at the same time.

Antibodies identified as 1B20 competitors in the above or other suitableassays may be tested for the ability to antagonize or neutralize (i)PCSK9 binding to the LDL receptor; and/or (ii) PCSK9 internalizationinto cells. These parameters may be measured through the use of assayssimilar to that employed or described in the current specification. Inspecific embodiments, the inhibition demonstrated by the competingantibody is at least about 10% inhibition. In other embodiments, theinhibition is at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 95%.

The present invention specifically encompasses PCSK9-specificantagonists and particularly monoclonal antibody molecules (and theircorresponding amino acid and nucleic acid sequences) that selectivelybind to the same epitope as 1B20 or an overlapping epitope interferingwith 1B20's binding to PCSK9. Monoclonal antibodies that specificallybind to the epitope of 1B20 or an overlapping epitope antagonize orneutralize (i) PCSK9 binding to the LDL receptor; (ii) PCSK9internalization into cells, or (iii) both. A monoclonal antibodymolecule in accordance herewith may be an intact (complete or fulllength) antibody, a substantially intact antibody, or a portion orfragment of an antibody comprising an antigen-binding portion, e.g., aFab fragment, Fab′ fragment or F(ab′)2 fragment of a murine antibody orof a chimeric antibody or of a humanized antibody or of a humanantibody. Monoclonal, as used herein, refers to a homogeneous orsubstantially homogeneous (or pure) antibody population (i.e., at leastabout 90%, 91%, 92%, 93%, 94%, 95%, 96%, more preferably at least about97% or 98%, or most preferably at least 99% of the antibodies in thepopulation are identical and would compete in an ELISA assay for thesame antigen or epitope. In specific embodiments of the presentinvention, the present invention provides monoclonal antibodies that (i)compete for binding to PCSK9 with a 1B20 antibody molecule, reducing1B20 binding by at least 50% at 1 μM or less with 1B20 at or below itsK_(D), (ii) block PCSK9 binding to the LDL receptor, (iii) inhibit PCSK9internalization into the cell, and (iv) comprise a specificantigen-binding region, VH, VL, set of CDRs or heavy CDR3, heavy and/orlight chain or any variant of these components described herein.

In any of the above assays for identifying antibodies binding the sameor overlapping epitope region as 1B20, binding of the known binder(i.e., 1B20 antibody molecule) as compared to the binding of thecandidate binder should be distinguishable. This can (but need not) beaccomplished through the use of labels on either or both molecules aswill be readily appreciated by the skilled artisan. Labels, as usedherein, refer to another molecule or agent incorporated into/affixed tothe antibody molecule. In one embodiment, the label is a detectablemarker, e.g., a radiolabeled amino acid or attachment to a polypeptideof biotinyl moieties that can be detected by marked avidin (e.g.,streptavidin containing a fluorescent marker or enzymatic activity thatcan be detected by optical or colorimetric methods). Various methods oflabeling polypeptides and glycoproteins are known in the art and may beused. Examples of labels for polypeptides include, but are not limitedto, the following: radioisotopes or radionuclides (e.g., ³H, ¹⁴C, ¹⁵N,³⁵S, ⁹⁰Y, ⁹⁹Tc, ¹¹¹In, ¹²⁵I, ¹³¹I), fluorescent labels (e.g., FITC,rhodamine, lanthanide phosphors), enzymatic labels (e.g., horseradishperoxidase, β-galactosidase, luciferase, alkaline phosphatase),chemiluminescent markers, biotinyl groups, predetermined polypeptideepitopes recognized by a secondary reporter (e.g., leucine zipper pairsequences, binding sites for secondary antibodies, metal bindingdomains, epitope tags), magnetic agents, such as gadolinium chelates,toxins such as pertussis toxin, taxol, cytochalasin B, gramicidin D,ethidium bromide, emetine, mitomycin, etoposide, tenoposide,vincristine, vinblastine, colchicin, doxorubicin, daunorubicin,dihydroxy anthracin dione, mitoxantrone, mithramycin, actinomycin D,1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine,propranolol, and puromycin and analogs or homologs thereof. In someembodiments, labels are attached by spacer arms of various lengths toreduce potential steric hindrance.

A 1B20 antibody used for the competition assays may be any antibodymolecule which is of the 1B20 description provided herein (i.e. anyantibody molecule selective for PCSK9 which comprises a VL comprisingSEQ ID NO: 27 and a VH comprising SEQ ID NO: 11). Examples of suchantibodies include without limitation (i) a Fab which comprises a lightchain comprising SEQ ID NO: 1 and an Fd chain comprising amino acids1-221 of SEQ ID NO: 9 (or SEQ ID NO: 9); (ii) a full length antibodymolecule which comprises a light chain comprising SEQ ID NO: 26 and aheavy chain comprising SEQ ID NO: 25.

Expression and selection of any of the PCSK9-specific antagonistsdescribed in the present application may be achieved using suitabletechnologies including, but not limited to phage display (see, e.g.,International Application Number WO 92/01047, Kay et al., 1996 PhageDisplay of Peptides and Proteins: A Laboratory Manual, San Diego:Academic Press), yeast display, bacterial display, T7 display, andribosome display (see, e.g., Lowe & Jermutus, 2004 Curr. Pharm. Biotech.517-527).

Particular PCSK9-specific antagonists forming part of the presentinvention are antibody molecules or antibodies. “Antibody molecule” or“Antibody” as described herein refers to an immunoglobulin-derivedstructure with selective binding to human and/or murine PCSK9 including,but not limited to, a full length or whole antibody, an antigen bindingfragment (a fragment derived, physically or conceptually, from anantibody structure), a derivative of any of the foregoing, a fusion ofany of the foregoing with another polypeptide, or any alternativestructure/composition which incorporates any of the foregoing forpurposes of selectively binding to/inhibiting the function of PCSK9.

“Whole” antibodies or “full length” antibodies refer to proteins thatcomprise two heavy (H) and two light (L) chains inter-connected bydisulfide bonds which comprise: (1) in terms of the heavy chains, avariable region (abbreviated herein as “V_(H)”) and a heavy chainconstant region which comprises three domains, C_(H1), C_(H2), andC_(H3); and (2) in terms of the light chains, a light chain variableregion (abbreviated herein as “V_(L)”) and a light chain constant regionwhich comprises one domain, C_(L).

Antibody fragments and, more specifically, antigen binding fragments aremolecules possessing an antibody variable region or segment thereof(which comprises one or more of the disclosed CDR 3 domains, heavyand/or light within framework regions of heavy and/or light chains, asappropriate), which confers selective binding to PCSK9, and particularlyhuman and/or murine PCSK9. Antibody fragments containing such anantibody variable region include, but are not limited to the followingantibody molecules: a Fab, a F(ab′)₂, a Fd, a Fv, a scFv, bispecificantibody molecules (antibody molecules comprising a PCSK9-specificantibody or antigen binding fragment as disclosed herein linked to asecond functional moiety having a different binding specificity than theantibody, including, without limitation, another peptide or protein suchas an antibody, or receptor ligand), a bispecific single chain Fv dimer,an isolated CDR3, a minibody, a ‘scAb’, a dAb fragment, a diabody, atriabody, a tetrabody, a minibody, and artificial antibodies based uponprotein scaffolds, including but not limited to fibronectin type IIIpolypeptide antibodies (see, e.g., U.S. Pat. No. 6,703,199 andInternational Application Numbers WO 02/32925 and WO 00/34784) orcytochrome B; see, e.g., Nygren et al., 1997 Curr. Opinion Struct. Biol.7:463-469; the disclosures of which are incorporated herein byreference. The antibody portions or binding fragments may be natural, orpartly or wholly synthetically produced. Such antibody portions can beprepared by various means known by one of skill in the art, including,but not limited to, conventional techniques, such as papain or pepsindigestion.

The term “isolated” as used herein in reference to antibody molecules,PCSK9-specific antagonists in general, encoding nucleic acid or otherdescribes a property as it pertains to the disclosed PCSK9-specificantagonists, nucleic acid or other that makes them different from thatfound in nature. The difference can be, for example, that they are of adifferent purity than that found in nature, or that they are of adifferent structure or form part of a different structure than thatfound in nature. A structure not found in nature, for example, includesrecombinant human immunoglobulin structures including, but not limitedto, recombinant human immunoglobulin structures with optimized CDRs.Other examples of structures not found in nature are PCSK9-specificantagonists or nucleic acid substantially free of other cellularmaterial. Isolated PCSK9-specific antagonists are generally free ofother protein-specific antagonists having different proteinspecificities (i.e., possess an affinity for other than PCSK9).

In one particular aspect, the present invention provides isolatedPCSK9-specific antagonists which antagonize PCSK9 function. Inparticular embodiments, said PCSK9-specific antagonists inhibit humanand/or murine PCSK9's antagonism of cellular LDL uptake by interferingwith PCSK9 binding to the LDL receptor and resultant PCSK9 cellinternalization. Disclosed PCSK9-specific antagonists, thus, formdesirable molecules for lowering plasma LDL-cholesterol levels; see,e.g., Cohen et al., 2005 Nat. Genet. 37:161-165 (wherein significantlylower plasma LDL cholesterol levels were noted in individualsheterozygous for a nonsense mutation in allele PCSK9); Rashid et al.,2005 Proc. Natl. Acad. Sci. USA 102:5374-5379 (wherein PCSK9-knockoutmice evidenced increased numbers of LDLRs in hepatocytes, acceleratedplasma LDL clearance, and significantly lower plasma cholesterollevels); and Cohen et al., 2006 N. Engl. J. Med. 354:1264-1272 (whereinhumans heterozygous for mutated, loss of function, PCSK9 exhibited asignificant reduction in the long-term risk of developingatherosclerotic heart disease).

Through repeat experiments, 1B20 antibody molecules as disclosed hereindose-dependently inhibited the effects of both human and/or murine PCSK9on LDL uptake. In specific embodiments, the present invention, thus,encompasses isolated PCSK9-specific antagonists and, in more specificembodiments, antibody molecules comprising the heavy and/or light chainvariable regions (SEQ ID NO: 11 and 27, respectively) contained withinthese 1B20 antibody molecules or the heavy and/or light chains, e.g.,amino acids 1-221 of SEQ ID NO: 9 (or SEQ ID NO: 9) and SEQ ID NO: 1,respectively, or SEQ ID NOs: 25 and 26, respectively, as well asequivalents (characterized as having one or more conservative amino acidsubstitutions that do not degrade the PCSK9-selective property of 1B20)or homologs thereof. Particular embodiments comprise isolatedPCSK9-specific antagonists that comprise the CDR domains disclosedherein or sets of heavy and/or light chain CDR domains disclosed herein,or equivalents thereof, characterized as having one or more conservativeamino acid substitutions.

Use of the terms “domain” or “region” herein simply refers to therespective portion of the antibody molecule wherein the sequence orsegment at issue will reside or, in the alternative, currently resides.

In specific embodiments, the present invention provides isolatedPCSK9-specific antagonists and, in more specific embodiments, antibodymolecules comprising a heavy chain variable region which comprises SEQID NO: 11; equivalents thereof characterized as having one or moreconservative amino acid substitutions, and homologs thereof. Thedisclosed antagonists should counteract or inhibit human and/or murinePCSK9-dependent inhibition of cellular LDL uptake. In specificembodiments, the present invention provides homologs of the disclosedantagonists characterized as being at least 90% identical over the heavychain variable region to SEQ ID NO: 11; said antagonists which inhibithuman and/or murine PCSK9-dependent inhibition of cellular LDL uptake byat least 10%.

In specific embodiments, the present invention provides isolatedPCSK9-specific antagonists and, in more specific embodiments, antibodymolecules comprising a light chain variable region which comprises SEQID NO: 27; equivalents thereof characterized as having one or moreconservative amino acid substitutions, and homologs thereof. Thedisclosed antagonists should counteract or inhibit human and/or murinePCSK9-dependent inhibition of cellular LDL uptake. In specificembodiments, the present invention provides homologs of the disclosedantagonists characterized as being at least 90% identical over the lightchain variable region to SEQ ID NO: 27; said antagonists which inhibithuman and/or murine PCSK9-dependent inhibition of cellular LDL uptake byat least 10%.

In specific embodiments, the present invention provides isolatedPCSK9-specific antibody molecules which comprise a heavy chain variableregion comprising SEQ ID NO: 11 and a light chain variable regioncomprising SEQ ID NO: 27; or equivalents thereof characterized as havingone or more conservative amino acid substitutions in the prescribedsequences. Specific embodiments are said antagonists which inhibit humanand/or murine PCSK9-dependent inhibition of cellular LDL uptake by atleast 10%. In specific embodiments, the present invention provideshomologs of the disclosed antagonists characterized as being at least90% identical over the heavy and light chain variable regions to SEQ IDNOs: 11 and 27, respectively; said antagonists which inhibit humanand/or murine PCSK9-dependent inhibition of cellular LDL uptake by atleast 10%.

In particular embodiments, the present invention provides isolatedPCSK9-specific antagonists and, in more specific embodiments, PCSK9antibody molecules that comprise variable heavy CDR3 sequence SEQ ID NO:17; and equivalents thereof characterized as having one or moreconservative amino acid substitutions; specific embodiments of whichinhibit human and/or murine PCSK9-dependent inhibition of cellular LDLuptake by at least 10%. Specific embodiments provide isolatedantagonists which additionally comprise in the heavy chain variableregion CDR1 and/or CDR2 sequences comprising SEQ ID NO: 13 and/or SEQ IDNO: 15, respectively; or equivalents thereof characterized as having oneor more conservative amino acid substitutions in any one or more of theCDR sequences. In specific embodiments, the present invention provideshomologs of the disclosed antagonists characterized as being at least90% identical over the CDR3 sequences or within each of the CDR1, CDR2and CDR3 sequences to SEQ ID NO: 17 or SEQ ID NOs: 13, 15 and 17,respectively, as appropriate; said antagonists which inhibit humanand/or murine PCSK9-dependent inhibition of cellular LDL uptake by atleast 10%.

In particular embodiments, the present invention provides isolatedPCSK9-specific antagonists and, in more specific embodiments, antibodymolecules which comprise variable light CDR3 sequence which comprisesSEQ ID NO: 7; and equivalents thereof characterized as having one ormore conservative amino acid substitutions; specific embodiments ofwhich inhibit human and/or murine PCSK9-dependent inhibition of cellularLDL uptake by at least 10%. Specific embodiments provide isolatedantagonists which additionally comprise in the light chain variableregion CDR1 and/or CDR2 sequences comprising SEQ ID NO: 3 and/or SEQ IDNO: 5, respectively; or an equivalent thereof characterized as havingone or more conservative amino acid substitutions in any one or more ofthe CDR sequences. In specific embodiments, the present inventionprovides homologs of the disclosed antagonists characterized as being atleast 90% identical over the CDR3 sequences or within each of the CDR1,CDR2 and CDR3 sequences to SEQ ID NO: 7 or SEQ ID NOs: 3, 5 and 7,respectively, as appropriate; said antagonists which inhibit humanand/or murine PCSK9-dependent inhibition of cellular LDL uptake by atleast 10%.

In particular embodiments, the present invention provides isolatedPCSK9-specific antagonists and, in more specific embodiments, antibodymolecules which comprise heavy chain variable region CDR3 sequence andlight chain variable region CDR3 sequence comprising SEQ ID NOs: 17 and7, respectively; or equivalents thereof characterized as having one ormore conservative amino acid substitutions in any one or more of theCDR3 sequences; specific embodiments of which inhibit human and/ormurine PCSK9-dependent inhibition of cellular LDL uptake by at least10%. In specific embodiments, the present invention provides homologs ofthe disclosed antagonists characterized as being at least 90% identicalover the heavy and light chain variable region CDR3 sequences to SEQ IDNOs: 17 and 7, respectively; said antagonists which inhibit human and/ormurine PCSK9-dependent inhibition of cellular LDL uptake by at least10%.

Specific embodiments provide isolated PCSK9-specific antagonists and, inmore specific embodiments, antibody molecules which comprise heavy chainvariable region CDR1, CDR2, and CDR3 sequences and light chain variableregion CDR1, CDR2, and CDR3 sequences comprising SEQ ID NOs: 13, 15, 17,3, 5 and 7, respectively; and equivalents thereof characterized ashaving one or more conservative amino acid substitutions in any one ormore of the CDR sequences; specific embodiments of which inhibit humanand/or murine PCSK9-dependent inhibition of cellular LDL uptake by atleast 10%. In specific embodiments, the present invention provideshomologs of the disclosed antagonists characterized as being at least90% identical over the heavy and light chain variable region CDR1, CDR2and CDR3 sequences to SEQ ID NOs: 13, 15, 17, 3, 5 and 7, respectively;said antagonists which inhibit human and/or murine PCSK9-dependentinhibition of cellular LDL uptake by at least 10%.

One particular aspect of the present invention encompasses isolatedPCSK9-specific antagonists and, in more specific embodiments, antibodymolecules which are variants of that disclosed above which comprise aheavy chain variable region CDR3 sequence of SEQ ID NO: 39 (or, inparticular embodiments, SEQ ID NO: 98) wherein the CDR3 sequence is notSEQ ID NO: 17; specific embodiments of which inhibit human and/or murinePCSK9-dependent inhibition of cellular LDL uptake by at least 10%.Further embodiments hereof additionally comprise heavy chain variableregion CDR1 sequence of SEQ ID NO: 37 wherein the variant sequence isnot SEQ ID NO: 13 and/or heavy chain variable region CDR2 sequence ofSEQ ID NO: 38 (or, in particular embodiments, SEQ ID NO: 97) wherein thevariant sequence is not SEQ ID NO: 15; specific embodiments of whichinhibit human and/or murine PCSK9-dependent inhibition of cellular LDLuptake by at least 10%. In other embodiments, the present inventionencompasses heavy chain variable region sequence comprising CDR1, CDR2,and CDR3 sequence which, respectively, comprises SEQ ID NOs: 37, 38 and39 (or in particular embodiments, SEQ ID NOs: 37, 97, and 98) in therespective regions, which are, respectively, not SEQ ID NOs: 13, 15 and17; specific embodiments of which inhibit human and/or murinePCSK9-dependent inhibition of cellular LDL uptake by at least 10%.

Another aspect of the present invention encompasses isolatedPCSK9-specific antagonists and, in more specific embodiments, antibodymolecules which are variants of that disclosed above which comprise alight chain variable region CDR3 sequence of SEQ ID NO: 42 (or, inparticular embodiments, SEQ ID NO: 101) wherein the CDR3 sequence is notSEQ ID NO: 7; specific embodiments of which inhibit human and/or murinePCSK9-dependent inhibition of cellular LDL uptake by at least 10%.Further embodiments hereof additionally comprise light chain variableregion CDR1 sequence of SEQ ID NO: 40 (or, in particular embodiments,SEQ ID NO: 99) wherein the variant sequence is not SEQ ID NO: 3 and/orlight chain variable region CDR2 sequence of SEQ ID NO: 41 (or, inparticular embodiments, SEQ ID NO: 100) wherein the variant sequence isnot SEQ ID NO: 5; specific embodiments of which inhibit human and/ormurine PCSK9-dependent inhibition of cellular LDL uptake by at least10%. In other embodiments, the present invention encompasses light chainvariable region sequence comprising CDR1, CDR2 and CDR3 sequence which,respectively, comprises SEQ ID NOs: 40, 41 and 42 (or, in particularembodiments, SEQ ID NOs: 99, 100 and 101) in the respective regions,which are, respectively, not SEQ ID NOs: 3, 5 and 7; specificembodiments of which inhibit human and/or murine PCSK9-dependentinhibition of cellular LDL uptake by at least 10%.

Additional distinct embodiments encompass isolated PCSK9-specificantagonists which comprise: (a) a heavy chain variable region comprisingCDR1, CDR2 and CDR3 sequence, wherein (i) the CDR1 sequence comprisesSEQ ID NO: 13 or SEQ ID NO: 37; SEQ ID NO: 37 being different insequence from SEQ ID NO: 13; (ii) the CDR2 sequence comprises SEQ ID NO:15, SEQ ID NO: 38 or SEQ ID NO: 97; SEQ ID NOs: 38 and 97 beingdifferent in sequence from SEQ ID NO: 15; and (iii) the CDR3 sequencecomprises SEQ ID NO: 17, SEQ ID NO: 39 or SEQ ID NO: 98; SEQ ID NO: 39and SEQ ID NO: 98 being different in sequence from SEQ ID NO: 17; and/or(b) a light chain variable region comprising CDR1, CDR2 and CDR3sequence, wherein (i) the CDR1 sequence comprises SEQ ID NO: 3 or SEQ IDNO: 40; SEQ ID NO: 40 being different in sequence from SEQ ID NO: 3;(ii) the CDR2 sequence comprises SEQ ID NO: 5, SEQ ID NO: 41 or SEQ IDNO: 100; SEQ ID NOs: 41 and 100 being different in sequence from SEQ IDNO: 5; and (iii) the CDR3 sequence comprises SEQ ID NO: 7, SEQ ID NO: 42or SEQ ID NO: 101; SEQ ID NO: 42 and SEQ ID NO: 101 being different insequence from SEQ ID NO: 7; specific embodiments of which inhibit humanand/or murine PCSK9-dependent inhibition of cellular LDL uptake by atleast 10%.

Other aspects of the present invention encompass isolated PCSK9-specificantagonists and, in more specific embodiments, antibody molecules whichare variants of that disclosed above which comprise (i) a heavy chainvariable region sequence comprising CDR1, CDR2, and CDR3 sequence which,respectively, comprises SEQ ID NOs: 37, 38 and 39 (or, in particularembodiments, SEQ ID NOs: 37, 97 and 98) in the respective regions, whichare, respectively, not SEQ ID NOs:13, 15 and 17; and (ii) a light chainvariable region sequence comprising CDR1, CDR2 and CDR3 sequence which,respectively, comprises SEQ ID NOs: 40, 41 and 42 (or, in particularembodiments, SEQ ID NOs: 99, 100 and 101) in the respective regions,which are, respectively, not SEQ ID NOs: 3, 5 and 7; specificembodiments of which inhibit human and/or murine PCSK9-dependentinhibition of cellular LDL uptake by at least 10%.

In specific embodiments herein the CDRs are in place of thecorresponding regions of 1B20 with out without conservative amino acidsubstitutions; specific embodiments of which inhibit human and/or murinePCSK9-dependent inhibition of cellular LDL uptake by at least 10%. Inparticular embodiments, the present invention encompasses isolatedPCSK9-specific antagonists and, in more specific embodiments, antibodymolecules comprising heavy and/or light chain variable regionscomprising SEQ ID NOs: 44 and 43 (or, in particular embodiments, SEQ IDNOs: 109 and 108), respectively; said variants SEQ ID NOs which are notSEQ ID NOs: 11 and 27, respectively; specific embodiments of whichinhibit human and/or murine PCSK9-dependent inhibition of cellular LDLuptake by at least 10%.

Specific embodiments include any isolated PCSK9-specific antagonist and,in more specific embodiments, antibody molecules which comprise heavychain variable region sequence found in any of SEQ ID NOs: 45-96 and102-107, optionally comprising a light chain variable region sequencedisclosed herein (e.g., SEQ ID NO: 27); specific embodiments of whichinhibit human and/or murine PCSK9-dependent inhibition of cellular LDLuptake by at least 10%.

Particular embodiments are isolated PCSK9-specific antagonists whichcomprise the above-described VH and VL regions in a full lengthantibody. Specific embodiments herein further comprise a series of aminoacids selected from the group consisting of: SEQ ID NO: 21 (IgG1), SEQID NO: 22 (IgG2), SEQ ID NO: 23 (IgG4) and SEQ ID NO: 24 (IgG2 m4).

Conservative amino acid substitutions, as one of ordinary skill in theart will appreciate, are substitutions that replace an amino acidresidue with one imparting similar or better (for the intended purpose)functional and/or chemical characteristics. Antagonists bearing suchconservative amino acid substitutions can be tested for retained orbetter activity using functional assays available in the art ordescribed herein. PCSK9-specific antagonists possessing one or moreconservative amino acid substitutions which retain the ability toselectively bind to human PCSK9 and antagonize PCSK9 functioning at alevel the same or better than 1B20 antibody molecules as describedherein are referred to herein as “functional equivalents” of thedisclosed antagonists and form specific embodiments of the presentinvention. Conservative amino acid substitutions are often ones in whichthe amino acid residue is replaced with an amino acid residue having asimilar side chain. Families of amino acid residues having similar sidechains have been defined in the art. These families include amino acidswith basic side chains (e.g., lysine, arginine, histidine), acidic sidechains (e.g., aspartic acid, glutamic acid), uncharged polar side chains(e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine,cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine,leucine, isoleucine, proline, phenylalanine, methionine), beta-branchedside chains (e.g., threonine, valine, isoleucine) and aromatic sidechains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Suchmodifications are not designed to significantly reduce or alter thebinding or functional inhibition characteristics of the PCSK9-specificantagonist, albeit they may improve such properties. The purpose formaking a substitution is not significant and can include, but is by nomeans limited to, replacing a residue with one better able to maintainor enhance the structure of the molecule, the charge or hydrophobicityof the molecule, or the size of the molecule. For instance, one maydesire simply to substitute a less desired residue with one of the samepolarity or charge. Such modifications can be introduced by standardtechniques known in the art, such as site-directed mutagenesis andPCR-mediated mutagenesis. One specific means by which those of skill inthe art accomplish conservative amino acid substitutions is alaninescanning mutagenesis as discussed in, for example, MacLennan et al.,1998 Acta Physiol. Scand. Suppl. 643:55-67, and Sasaki et al., 1998 Adv.Biophys. 35:1-24.

In another aspect, the present invention provides isolatedPCSK9-specific antagonists and, in more specific embodiments, antibodymolecules which comprise heavy and/or light chain variable regionscomprising amino acid sequences that are homologous to the correspondingamino acid sequences of the disclosed antibodies, wherein the antibodymolecules inhibit PCSK9-dependent inhibition of cellular LDL uptake.Specific embodiments are antagonists which comprise heavy and/or lightchain variable regions which are at least 90% identical to disclosedheavy and/or light chain variable regions, respectively. Reference to“at least 90% identical” includes at least 90, 91, 92, 93, 94, 95, 96,97, 98, 99 and 100% identical sequences along the full length of themolecule disclosed herein.

PCSK9-specific antagonists with amino acid sequences homologous to theamino acid sequences of antagonists described herein are typicallyproduced to improve one or more of the properties of the antagonistwithout negatively impacting its specificity for PCSK9. One method ofobtaining such sequences, which is not the only method available to theskilled artisan, is to mutate sequence encoding the PCSK9-specificantagonist or specificity-determining region(s) thereof, express anantagonist comprising the mutated sequence(s), and test the encodedantagonist for retained function using available functional assaysincluding those described herein. Mutation may be by site-directed orrandom mutagenesis. As one of skill in the art will appreciate, however,other methods of mutagenesis can readily bring about the same effect.For example, in certain methods, the spectrum of mutants are constrainedby non-randomly targeting conservative substitutions based on eitheramino acid chemical or structural characteristics, or else by proteinstructural considerations. In affinity maturation experiments, severalsuch mutations may be found in a single selected molecule, whether theyare randomly or non-randomly selected. There are also variousstructure-based approaches toward affinity maturation as demonstratedin, e.g., U.S. Pat. No. 7,117,096, PCT Pub. Nos.: WO 02/084277 and WO03/099999; the disclosures of which are incorporated herein byreference.

As used herein, the percent homology between two amino acid or nucleicacid sequences is equivalent to the percent identity between the twosequences, and these two terms will be used interchangeably throughout.As used herein, % identity of two nucleic acid or amino acid sequencesis determined using the algorithm of Karlin and Altschul (Proc. Natl.Acad. Sci. USA 90:5873-5877, 1993). Such an algorithm is incorporatedinto the NBLAST and XBLAST programs of Altschul et al., 1990 J. Mol.Biol. 215:403-410. BLAST nucleotide searches are performed with theNBLAST program, score=100, wordlength=12, to obtain nucleic acidsequences homologous to a nucleic acid molecule of the invention. BLASTprotein searches are performed with the XBLAST program, score=50,wordlength=3, to obtain amino acid sequences homologous to an amino acidsequence disclosed herein. To obtain gapped alignments for comparisonpurposes, Gapped BLAST is utilized as described in Altschul et al., 1997Nucleic Acids Res. 25:3389-3402. When utilizing BLAST and Gapped BLASTprograms, the default parameters of the respective programs (e.g.,XBLAST and NBLAST) are used.

Utilization of components of one or more disclosed PCSK9-specificmolecules to produce other binding molecules with similar or betterspecificity is well within the realm of one skilled in the art. This canbe accomplished, for example, using techniques of recombinant DNAtechnology. One specific example of this involves the introduction ofDNA encoding the immunoglobulin variable region, or one or more of theCDRs, of an antibody to the variable region, constant region, orconstant region plus framework regions, as appropriate, of a differentimmunoglobulin. Such molecules form important aspects of the presentinvention. Specific immunoglobulins or the corresponding sequences, intowhich particular disclosed sequences may be inserted or, in thealternative, form the essential part of, include but are not limited tothe following antibody molecules which form particular embodiments ofthe present invention: a Fab (monovalent fragment with variable light(VL), variable heavy (VH), constant light (CL) and constant heavy 1(CH1) domains), a F(ab′)2 (bivalent fragment comprising two Fabfragments linked by a disulfide bridge or alternative at the hingeregion), a Fd (VH and CH1 domains), a Fv (VL and VH domains), a scFv (asingle chain Fv where VL and VH are joined by a linker, e.g., a peptidelinker, see, e.g., Bird et al., 1988 Science 242:423-426, Huston et al.,1988 PNAS USA 85:5879 -5883), a bispecific antibody molecule (anantibody molecule comprising a PCSK9-specific antibody or antigenbinding fragment as disclosed herein linked to a second functionalmoiety having a different binding specificity than the antibody,including, without limitation, another peptide or protein such as anantibody, or receptor ligand), a bispecific single chain Fv dimer (see,e.g., PCT/US92/09965), an isolated CDR3, a minibody (single chain-CH3fusion that self assembles into a bivalent dimer of about 80 kDa), a‘scAb’ (an antibody fragment containing VH and VL as well as either CLor CH1), a dAb fragment (VH domain, see, e.g., Ward et al., 1989 Nature341:544-546, and McCafferty et al., 1990 Nature 348:552-554; or VLdomain; Holt et al., 2003 Trends in Biotechnology 21:484-489), a diabody(see, e.g., Holliger et al., 1993 PNAS USA 90:6444-6448 andInternational Application Number WO 94/13804), a triabody, a tetrabody,a minibody (a scFv joined to a CH3; see, e.g., Hu et al., 1996 CancerRes. 56:3055-3061), IgG, IgG1, IgG2, IgG3, IgG4, IgM, IgD, IgA, IgE orany derivatives thereof, and artificial antibodies based upon proteinscaffolds, including but not limited to fibronectin type III polypeptideantibodies (see, e.g., U.S. Pat. No. 6,703,199 and InternationalApplication Number WO 02/32925) or cytochrome B; see, e.g., Koide etal., 1998 J. Molec. Biol. 284:1141-1151, and Nygren et al., 1997 CurrentOpinion in Structural Biology 7:463-469; the disclosures of which areincorporated herein by reference. Certain antibody molecules including,but not limited to, Fv, scFv, diabody molecules or domain antibodies(Domantis) may be stabilized by incorporating disulfide bridges to linethe VH and VL domains, see, e.g., Reiter et al., 1996 Nature Biotech.14:1239-1245; the disclosure of which is incorporated herein byreference. Bispecific antibodies may be produced using conventionaltechnologies (see, e.g., Holliger & Winter, 1993 Current OpinionBiotechnol. 4:446-449, specific methods of which include productionchemically, or from hybrid hybridomas) and other technologies including,but not limited to, the BiTE™ technology (molecules possessing antigenbinding regions of different specificity with a peptide linker) andknobs-into-holes engineering (see, e.g., Ridgeway et al., 1996 ProteinEng. 9:616-621; the disclosure of which is incorporated herein byreference). Bispecific diabodies may be produced in E. coli, and thesemolecules as other PCSK9-specific antagonists, as one of skill in theart will appreciate, may be selected using phage display in theappropriate libraries (see, e.g., International Application Number WO94/13804; the disclosure of which is incorporated herein by reference).

Variable domains, into which CDRs of interest are inserted, may beobtained from any germ-line or rearranged human variable domain.Variable domains may also be synthetically produced. The CDR regions canbe introduced into the respective variable domains using recombinant DNAtechnology. One means by which this can be achieved is described inMarks et al., 1992 Bio/Technology 10:779-783; the disclosure of which isincorporated herein by reference. A variable heavy domain may be pairedwith a variable light domain to provide an antigen binding site. Inaddition, independent regions (e.g., a variable heavy domain alone) maybe used to bind antigen. The artisan is well aware, as well, that twodomains of an Fv fragment, VL and VH, while perhaps coded by separategenes, may be joined, using recombinant methods, by a synthetic linkerthat enables them to be made as a single protein chain in which the VLand VH regions pair to form monovalent molecules (scFvs).

Specific embodiments provide the CDR(s) in germline framework regions.Framework regions, including but not limited to human framework regions,are known to those of skill in the art (e.g., a human or non-humanframework). The framework regions may be naturally occurring orconsensus framework regions. In one aspect, the framework region of anantibody of the invention is human (see, e.g., Clothia et al., 1998 J.Mol. Biol. 278:457-479 for a listing of human framework regions; saiddisclosure of which is incorporated herein by reference in itsentirety). Specific embodiments herein provide heavy chain variable CDR3SEQ ID NO: 17 into VH5_(—)3 in place of the relevant CDR. Specificembodiments herein provide heavy chain variable CDR1, CDR2 and/or CDR3sequences (SEQ ID NO:s 13, 15 and 17, respectively) into VH5_(—)3 inplace of the relevant CDRs. Specific embodiments herein provide lightchain variable CDR3 SEQ ID NO: 7 into VK4_(—)3 in place of the relevantCDR. Specific embodiments herein provide light chain variable CDR1, CDR2and/or CDR3 sequences (SEQ ID NO:s 3, 5 and 7, respectively) intoVK4_(—)3 in place of the relevant CDRs. Specific embodiments furtherprovide heavy chain variable CDR3SEQ ID NO: 17 and light chain variableCDR3SEQ ID NO: 7 into VH5_(—)3 and VK4_(—)3 germline sequences,respectively. Further embodiments provide heavy chain variable CDR1,CDR2 and/or CDR3 sequences (SEQ ID NO:s 13, 15 and 17, respectively)into VH5_(—)3 in place of the relevant CDRs; and light chain variableCDR1, CDR2 and/or CDR3 sequences (SEQ ID NO:s 3, 5 and 7, respectively)into VK4_(—)3 in place of the relevant CDRs.

The present invention encompasses antibody molecules that are human,humanized, deimmunized, chimeric and primatized. The invention alsoencompasses antibody molecules produced by the process of veneering;see, e.g., Mark et al., 1994 Handbook of Experimental Pharmacology, vol.113: The pharmacology of monoclonal Antibodies, Springer-Verlag, pp.105-134; the disclosure of which is incorporated herein by reference.“Human” in reference to the disclosed antibody molecules specificallyrefers to antibody molecules having variable and/or constant regionsderived from human germline immunoglobulin sequences, wherein saidsequences may, but need not, be modified/altered to have certain aminoacid substitutions or residues that are not encoded by human germlineimmunoglobulin sequence. Such mutations can be introduced by methodsincluding, but not limited to, random or site-specific mutagenesis invitro, or by somatic mutation in vivo. Specific examples of mutationtechniques discussed in the literature are that disclosed in Gram etal., 1992 PNAS USA 89:3576-3580; Barbas et al., 1994 PNAS USA91:3809-3813, and Schier et al., 1996 J. Mol. Biol. 263:551-567; thedisclosures of which are incorporated herein by reference. These areonly specific examples and do not represent the only availabletechniques. There are a plethora of mutation techniques in thescientific literature which are available to, and widely appreciated by,the skilled artisan. “Humanized” in reference to the disclosed antibodymolecules refers specifically to antibody molecules wherein CDRsequences derived from another mammalian species, such as a mouse, aregrafted onto human framework sequences. “Primatized” in reference to thedisclosed antibody molecules refers to antibody molecules wherein CDRsequences of a non-primate are inserted into primate frameworksequences, see, e.g., WO 93/02108 and WO 99/55369; the disclosures ofwhich are incorporated herein by reference.

Specific antibodies of the present invention are monoclonal antibodiesand, in particular embodiments, are in one of the following antibodyformats: IgD, IgA, IgE, IgM, IgG 1, IgG2, IgG3, IgG4 or any derivativeof any of the foregoing. The language “derivatives thereof” or“derivatives” in this respect includes, inter alia, (i) antibodies andantibody molecules with conservative modifications in one or bothvariable regions (i.e., VH and/or VL), (ii) antibodies and antibodymolecules with manipulations in the constant regions of the heavy and/orlight chains, and/or (iii) antibodies and antibody molecules thatcontain additional chemical moieties which are not normally a part ofthe immunoglobulin molecule (e.g., pegylation).

Manipulations of the variable regions can be within one or more of theVH and/or VL CDR regions. Site-directed mutagenesis, random mutagenesisor other method for generating sequence or molecule diversity can beutilized to create mutants which can subsequently be tested for aparticular functional property of interest in available in vitro or invivo assays including those described herein.

Antibodies of the present invention also include those in whichmodifications have been made to the framework residues within VH and/orVL to improve one or more properties of the antibody of interest.Typically, such framework modifications are made to decrease theimmunogenicity of the antibody. For example, one approach is to“backmutate” one or more framework residues to the correspondinggermline sequence. More specifically, an antibody that has undergonesomatic mutation may contain framework residues that differ from thegermline sequence from which the antibody is derived. Such residues canbe identified by comparing the antibody framework sequences to thegermline sequences from which the antibody is derived. Such“backmutated” antibodies are also intended to be encompassed by theinvention. Another type of framework modification involves mutating oneor more residues within the framework region, or even within one or moreCDR regions, to remove T cell epitopes to thereby reduce the potentialimmunogenicity of the antibody. This approach is also referred to as“deimmunization” and is described in further detail in U.S. PatentPublication No. 20030153043 by Carr et al; the disclosure of which isincorporated herein by reference.

In addition or alternative to modifications made within the framework orCDR regions, antibodies of the invention may be engineered to includemodifications within the Fc or constant regions, where present,typically to alter one or more functional properties of the antibody,such as serum half-life, complement fixation, Fc receptor binding,and/or antigen-dependent cellular cytotoxicity.

The concept of generating “hybrids” or “combinatorial” IgG formscomprising various antibody isotypes to hone in on desired effectorfunctionality has generally been described; see, e.g., Tao et al., 1991J. Exp. Med. 173:1025-1028. A specific embodiment of the presentinvention encompasses antibody molecules that possess specificmanipulations in the Fc region which have been found to result inreduced or altered binding to FcγR receptors, C1q or FcRn on the part ofthe antibody. The present invention, therefore, encompasses antibodiesin accordance with the present description that do not provoke (orprovoke to a lesser extent) antibody-dependent cellular cytotoxicity(“ADCC”), complement-mediated cytotoxicity (“CMC”), or form immunecomplexes, while retaining normal pharmacokinetic (“PK”) properties.Specific embodiments of the present invention provide an antibodymolecule as defined in accordance with the present invention whichcomprises, as part of its immunoglobulin structure, SEQ ID NO: 24 and,in particular embodiments, residues 107-326 of SEQ ID NO: 24 as part ofthe immunoglobulin structure. The present invention encompasses antibodymolecules which comprise: (i) a light chain comprising SEQ ID NO: 1, and(ii) a heavy chain comprising SEQ ID NO: 11 in sequence with (adjacentto) or followed by a series of amino acids selected from the groupconsisting of: SEQ ID NO: 21 (IgG1), SEQ ID NO: 22 (IgG2), SEQ ID NO: 23(IgG4) and SEQ ID NO: 24 (IgG2 m4). FIG. 6 illustrates a comparison ofsequence comprising SEQ ID NO: 24, particularly IgG2 m4, with IgG1,IgG2, and IgG4. Amino acid sequences for mature, secreted anti-PCSK9IgG2m4 heavy and light chains can be found as SEQ ID NOs: 25 and 26,respectively. Antibody molecules encoded at least in part by saidsequence are encompassed herein.

Specific PCSK9-specific antagonists may carry a detectable label, or maybe conjugated to a toxin (e.g., a cytotoxin), a radioactive isotope, aradionuclide, a liposome, a targeting moiety, a biosensor, a cationictail, or an enzyme (e.g., via a peptidyl bond or linker). SuchPCSK9-specific antagonist compositions form an additional aspect of thepresent invention.

In another aspect, the present invention provides isolated nucleic acidencoding disclosed PCSK9-specific antagonists. “Isolated” as mentionedprior refers to the property of the thing referred to that makes themdifferent from that found in nature. The difference can be, for example,that they are of a different purity than that found in nature, or thatthey are of a different structure or form part of a different structurethan that found in nature. An example of nucleic acid not found innature is, for example, nucleic acid substantially free of othercellular material. The nucleic acid may be present in whole cells, in acell lysate, or in a partially purified or substantially pure form. Inspecific instances, a nucleic acid may be isolated when purified awayfrom other cellular components or other contaminants, e.g., othercellular nucleic acids or proteins, for example, using standardtechniques, including without limitation, alkaline/SDS treatment, CsClbanding, column chromatography, agarose gel electrophoresis and othersuitable methods known in the art. The nucleic acid may include DNA(inclusive of cDNA) and/or RNA. Nucleic acids of the present inventioncan be obtained using standard molecular biology techniques. Forantibodies expressed by hybridomas (e.g., hybridomas prepared fromtransgenic mice carrying human immunoglobulin genes), cDNAs encoding thelight and heavy chains of the antibody made by the hybridoma can beobtained by standard PCR amplification or cDNA cloning techniques. Forantibodies obtained from an immunoglobulin gene library (e.g., usingphage display techniques), nucleic acid encoding the antibody can berecovered from the library.

The present invention encompasses isolated nucleic acid encodingdisclosed variable heavy and/or light chains and select componentsthereof, particularly the disclosed variable or respective CDR regionsand, in particular CDR3. In specific embodiments hereof, the CDR(s) areprovided within antibody framework regions and, in particularembodiments, human framework regions. Specific embodiments provideisolated nucleic acid encoding the CDR(s) into germline frameworkregions including, but not limited to, human germline framework regions.Specific embodiments herein provide isolated nucleic acid encoding heavychain CDR SEQ ID NO: 17 (in specific embodiments, said nucleic acid ofwhich comprises SEQ ID NO: 18) into VH5_(—)3 in place of the nucleicacid encoding the relevant CDR. Specific embodiments herein providenucleic acid encoding heavy chain variable CDR1, CDR2 and/or CDR3sequences SEQ ID NOs: 13, 15 and 17, respectively (and, in particularembodiments, said nucleic acid of which comprises SEQ ID NOs: 14, 16 and18, respectively) into VH5_(—)3 in place of the relevant CDRs. Specificembodiments herein provide isolated nucleic encoding light chain CDR SEQID NO: 7 (in specific embodiments, said nucleic acid of which comprisesSEQ ID NO: 8) into VK4_(—)3 in place of the nucleic acid encoding therelevant CDR. Specific embodiments herein provide nucleic acid encodinglight chain variable CDR1, CDR2 and/or CDR3 sequences SEQ ID NOs: 3, 5and 7, respectively (and, in particular embodiments, said nucleic acidof which comprises SEQ ID NOs: 4, 6 and 8, respectively) into VK4_(—)3in place of the relevant CDRs. Specific embodiments further provideheavy chain variable CDR3 SEQ ID NO: 17 (and, in particular embodiments,said nucleic acid of which comprises SEQ ID NO: 18) and light chainvariable CDR3 SEQ ID NO: 7 (and, in particular embodiments, said nucleicacid of which comprises SEQ ID NO: 8) into VH5_(—)3 and VK4_(—)3germline sequences, respectively. Further embodiments provide heavychain variable CDR1, CDR2 and/or CDR3 sequences SEQ ID NOs: 13, 15 and17, respectively (and, in particular embodiments, said nucleic acid ofwhich comprises SEQ ID NOs: 14, 16 and 18, respectively) into VH5_(—)3in place of the relevant CDRs; and light chain variable CDR1, CDR2and/or CDR3 sequences SEQ ID NOs: 3, 5 and 7, respectively (and, inparticular embodiments, said nucleic acid of which comprises SEQ ID NOs:4, 6 and 8, respectively) into VK4_(—)3 in place of the relevant CDRs.

The isolated nucleic acid encoding the variable regions can be providedwithin any desired antibody molecule format including, but not limitedto, the following: F(ab′)₂, a Fab, a Fv, a scFv, bispecific antibodymolecules (antibody molecules comprising a PCSK9-specific antibody orantigen binding fragment as disclosed herein linked to a secondfunctional moiety having a different binding specificity than theantibody, including, without limitation, another peptide or protein suchas an antibody, or receptor ligand), a bispecific single chain Fv dimer,a minibody, a dAb fragment, diabody, triabody or tetrabody, a minibody,IgG, IgG1, IgG2, IgG3, IgG4, IgM, IgD, IgA, IgE or any derivativesthereof.

Specific embodiments provide isolated nucleic acid which encodesPCSK9-specific antagonists and, in more specific embodiments, antibodymolecules comprising a heavy chain variable domain which comprises SEQID NO: 11; specific embodiments of which comprise nucleic acid sequenceSEQ ID NO: 12. Specific embodiments of the present invention provideisolated nucleic acid encoding PCSK9-specific antagonists and, in morespecific embodiments, antibody molecules, which additionally comprise:(i) nucleic acid encoding heavy chain CDR1 amino acid sequence SEQ IDNO: 13 (specific embodiments of which comprise nucleic acid SEQ ID NO:14) and/or (ii) nucleic acid encoding heavy chain CDR2 amino acidsequence SEQ ID NO: 15 (specific embodiments of which comprise nucleicacid SEQ ID NO: 16). Specific embodiments provide isolated nucleic acidencoding PCSK9-specific antagonists and, in more specific embodiments,antibody molecules comprising a light chain variable domain whichcomprises SEQ ID NO: 27; specific embodiments of which comprise nucleicacid sequence SEQ ID NO: 28. Specific embodiments of the presentinvention provide isolated nucleic acid encoding PCSK9-specificantagonists and, in more specific embodiments, antibody molecules, whichadditionally comprise: (i) nucleic acid encoding light chain CDR1 aminoacid sequence SEQ ID NO: 3 (specific embodiments of which comprisenucleic acid SEQ ID NO: 4) and/or (ii) nucleic acid encoding light chainCDR2 amino acid sequence SEQ ID NO: 5 (specific embodiments of whichcomprise nucleic acid SEQ ID NO: 6). Specific embodiments provideisolated nucleic acid encoding PCSK9-specific antagonists and, in morespecific embodiments, antibody molecules which comprise a heavy chainvariable domain which comprises SEQ ID NO: 11; specific embodiments ofwhich comprise nucleic acid sequence SEQ ID NO: 12; and a light chainvariable domain which comprises SEQ ID NO: 27; specific embodiments ofwhich comprise nucleic acid sequence SEQ ID NO: 28. Specific embodimentsprovide isolated nucleic acid encoding (i) heavy chain CDR1, CDR2 and/orCDR3 sequences (SEQ ID NOs: 13, 15 and 17, respectively; specificembodiments of which comprise nucleic acid SEQ ID NOs: 14, 16 and/or 18,respectively) preferably in a framework region (including but notlimited to a human framework region); and (ii) light chain CDR1, CDR2and/or CDR3 sequences (SEQ ID NO: 3, 5 and 7, respectively; specificembodiments of which comprise nucleic acid SEQ ID NOs: 4, 6 and/or 8,respectively) preferably in a framework region (including but notlimited to a human framework region). The present invention furtherprovides in specific embodiments, homologs of the antagonists disclosedabove, characterized as being at least 90% identical over the heavyand/or light chain variable regions, or the CDR regions, as appropriate,whichever is present to the corresponding sequences of 1B20.

Additional embodiments provide isolated nucleic acid encodingPCSK9-specific antagonists and, in more specific embodiments, antibodymolecules which comprise a light chain comprising SEQ ID NO: 1 (specificembodiments of which comprise nucleic acid SEQ ID NO: 2) and a heavychain or Fd chain comprising amino acids 1-221 of SEQ ID NO: 9, or SEQID NO: 9 (specific embodiments of which comprise nucleic acid 1-663 ofSEQ ID NO: 10, or SEQ ID NO: 10; respectively). Further embodimentsprovide isolated nucleic acid encoding PCSK9-specific antagonists and,in more specific embodiments, antibody molecules which comprise a lightchain comprising SEQ ID NO: 26 (specific embodiments of which compriseSEQ ID NO: 30) and a heavy chain comprising SEQ ID NO: 25 (specificembodiments of which comprise SEQ ID NO: 29). The present inventionfurther provides in specific embodiments, homologs of the antagonistsdisclosed above, characterized as being at least 90% identical over theheavy and/or light chains to the corresponding sequences of 1B20.

Specific embodiments of the present invention encompass nucleic acidencoding antibody molecules that possess manipulations in the Fc regionwhich result in reduced or altered binding to FcγR receptors, C1 q orFcRn on the part of the antibody. One specific embodiment of the presentinvention is isolated nucleic acid which encodes for antibody moleculescomprising as part of their immunoglobulin structure SEQ ID NO: 24 and,in particular embodiments, residues 107-326 of SEQ ID NO: 24. Inspecific embodiments, synthetic PCSK9-specific antagonists can beproduced by expression from nucleic acid generated from oligonucleotidessynthesized and assembled within suitable expression vectors; see, e.g.,Knappick et al., 2000 J. Mol. Biol. 296:57-86, and Krebs et al., 2001 J.Immunol. Methods 254:67-84.

The present invention encompasses nucleic acid encoding antibodymolecules which comprise: (i) nucleic acid encoding a light chaincomprising SEQ ID NO: 1 (specific embodiments of which comprise nucleicacid SEQ ID NO: 2), and (ii) nucleic acid encoding a heavy chaincomprising SEQ ID NO: 11 (specific embodiments of which comprise nucleicacid SEQ ID NO: 12) followed in sequence by (adjacent to) a set ofnucleotides encoding for a set of amino acids selected from the groupconsisting of: SEQ ID NO: 21 (IgG1), SEQ ID NO: 22 (IgG2), SEQ ID NO: 23(IgG4) and SEQ ID NO: 24 (IgG2 m4). Nucleotide sequences for mature,secreted anti-PCSK9 IgG2m4 heavy and light chains can be found as SEQ IDNOs: 29 and 30, respectively. Plasmid sequences comprising heavy andlight chain 1B20 anti-PCSK9 IgG2m4 antibody molecules can be found asSEQ ID NOs: 35 and 36, respectively. Nucleic acid encoding such antibodymolecules form important embodiments hereof.

Also included within the present invention are isolated nucleic acidscomprising nucleotide sequences which are at least about 90% identicaland more preferably at least about 95% identical to the full length ofthe nucleotide sequences described herein, and which nucleotidesequences encode PCSK9-specific antagonists which inhibitPCSK9-dependent inhibition of cellular LDL uptake by at least 10%.

Reference to “at least about 90% identical” throughout the applicationincludes at least about 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100%identical.

The invention further provides isolated nucleic acid at least a portionof which hybridizes to the complement of nucleic acid consisting of SEQID NO: 12 and/or SEQ ID NO: 28 under stringent hybridization conditions,said nucleic acid of which confers upon antibody molecules the abilityto specifically bind PCSK9 and antagonize PCSK9 function, andPCSK9-specific antagonists expressed employing said nucleic acid.Methods for hybridizing nucleic acids are well-known in the art; see,e.g., Ausubel, Current Protocols in Molecular Biology, John Wiley &Sons, N.Y., 6.3.1-6.3.6, 1989. Stringent hybridization conditionsinvolve hybridizing at 68° C. in 5×SSC/5×Denhardt's solution (orequivalent)/1.0% SDS, and washing in 0.2×SSC/0.1% SDS at roomtemperature. Moderately stringent conditions include washing in 3×SSC at42° C. The parameters of salt concentration and temperature can bevaried to achieve the optimal level of identity between the probe andthe target nucleic acid. The skilled artisan can manipulate varioushybridization and/or washing conditions to specifically target nucleicacid in the hybridizing portion that is at least 80, 85, 90, 95, 98, or99% identical to SEQ ID NO: 12 and/or SEQ ID NO: 28. Basic parametersaffecting the choice of hybridization conditions and guidance fordevising suitable conditions are set forth by Sambrook et al., MolecularCloning. A Laboratory Manual, Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y., chapters 9 and 11, 1989 and Ausubel et al. (eds),Current Protocols in Molecular Biology, John Wiley & Sons, Inc.,sections 2.10 and 6.3-6.4, 1995 (the disclosures of which areincorporated herein by reference), and can be readily determined bythose having ordinary skill in the art. PCSK9 antagonists having one ormore variable regions comprising nucleic acid which hybridizes to thecomplement of nucleic acid consisting of SEQ ID NO: 12 and/or SEQ ID NO:28 under stringent hybridization conditions should be effective inantagonizing one or more functions of PCSK9. Said antagonists andencoding nucleic acid, thus, form important embodiments of the presentinvention.

In another aspect, the present invention provides vectors comprising thenucleic acid disclosed herein. Vectors in accordance with the presentinvention include, but are not limited to, plasmids and other expressionconstructs (e.g., phage or phagemid, as appropriate) suitable for theexpression of the desired antibody molecule at the appropriate level forthe intended purpose; see, e.g., Sambrook & Russell, Molecular Cloning:A Laboratory Manual: 3^(rd) Edition, Cold Spring Harbor LaboratoryPress; the disclosure of which is incorporated herein by reference. Formost cloning purposes, DNA vectors may be used. Typical vectors includeplasmids, modified viruses, bacteriophage, cosmids, yeast artificialchromosomes, bacterial artificial chromosomes, and other forms ofepisomal or integrated DNA. It is well within the purview of the skilledartisan to determine an appropriate vector for a particular genetransfer, generation of a recombinant PCSK9-specific antagonist, orother use. In specific embodiments, in addition to a recombinant gene,the vector may also contain an origin of replication for autonomousreplication in a host cell, appropriate regulatory sequences, such as apromoter, a termination sequence, a polyadenylation sequence, anenhancer sequence, a selectable marker, a limited number of usefulrestriction enzyme sites, and/or other sequences as appropriate and thepotential for high copy number. Examples of expression vectors for theproduction of protein-specific antagonists are well known in the art;see, e.g., Persic et al., 1997 Gene 187:9-18; Boel et al., 2000 J.Immunol. Methods 239:153-166, and Liang et al., 2001 J. Immunol. Methods247:119-130; the disclosures of which are incorporated herein byreference. If desired, nucleic acid encoding the antagonist may beintegrated into the host chromosome using techniques well known in theart; see, e.g., Ausubel, Current Protocols in Molecular Biology, JohnWiley & Sons, 1999, and Marks et al., International Application NumberWO 95/17516. Nucleic acid may also be expressed on plasmids maintainedepisomally or incorporated into an artificial chromosome; see, e.g.,Csonka et al., 2000 J. Cell Science 113:3207-3216; Vanderbyl et al.,2002 Molecular Therapy 5:10. Specifically with regards to antibodymolecules, the antibody light chain gene and the antibody heavy chaingene can be inserted into separate vectors or, more typically, bothgenes may be inserted into the same expression vector. Nucleic acidencoding any PCSK9-specific antagonist or component thereof can beinserted into an expression vector using standard methods (e.g.,ligation of complementary restriction sites on the nucleic acid fragmentand vector, or blunt end ligation if no restriction sites are present).Another specific example of how this may be carried out is through useof recombinational methods, e.g. the Clontech “InFusion” system, orInvitrogen “TOPO” system (both in vitro), or intracellularly (e.g. theCre-Lox system). Specifically with regards to antibody molecules, thelight and heavy chain variable regions can be used to create full-lengthantibody genes of any antibody isotype by inserting them into expressionvectors already encoding heavy chain constant and light chain constantregions of the desired isotype such that the VH segment is operativelylinked to the CH segment(s) within the vector and the VL segment isoperatively linked to the CL segment within the vector. Additionally oralternatively, the recombinant expression vector comprising nucleic acidencoding a PCSK9-specific antagonist can encode a signal peptide thatfacilitates secretion of the antagonist from a host cell. The nucleicacid can be cloned into the vector such that the nucleic acid encoding asignal peptide is linked in-frame adjacent to the PCSK9-specificantagonist-encoding nucleic acid. The signal peptide may be animmunoglobulin or a non-immunoglobulin signal peptide. Any techniqueavailable to the skilled artisan may be employed to introduce thenucleic acid into the host cell; see, e.g., Morrison, 1985 Science,229:1202. Methods of subcloning nucleic acid molecules of interest intoexpression vectors, transforming or transfecting host cells containingthe vectors, and methods of making substantially pure protein comprisingthe steps of introducing the respective expression vector into a hostcell, and cultivating the host cell under appropriate conditions arewell known. The PCSK9-specific antagonist so produced may be harvestedfrom the host cells in conventional ways. Techniques suitable for theintroduction of nucleic acid into cells of interest will depend on thetype of cell being used. General techniques include, but are not limitedto, calcium phosphate transfection, DEAE-Dextran, electroporation,liposome-mediated transfection and transduction using virusesappropriate to the cell line of interest (e.g., retrovirus, vaccinia,baculovirus, or bacteriophage).

In another aspect, the present invention provides isolated cell(s)comprising nucleic acid encoding disclosed PCSK9-specific antagonists. Avariety of different cell lines are contemplated herein and can be usedfor the recombinant production of PCSK9-specific antagonists, includingbut not limited to those from prokaryotic organisms (e.g., E. coli,Bacillus, and Streptomyces) and from eukaryotic (e.g., yeast,Baculovirus, and mammalian); see, e.g., Breitling et al., Recombinantantibodies, John Wiley & Sons, Inc. and Spektrum Akademischer Verlag,1999; the disclosure of which is incorporated herein by reference. Plantcells, including transgenic plants, and animal cells, includingtransgenic animals (other than humans), comprising the nucleic acid orantagonists disclosed herein are also contemplated as part of thepresent invention. Suitable mammalian cells or cell lines including, butnot limited to, those derived from Chinese Hamster Ovary (CHO cells,including but not limited to DHFR-CHO cells (described in Urlaub andChasin, 1980 Proc. Natl. Acad. Sci. USA 77:4216-4220) used, for example,with a DHFR selectable marker (e.g., as described in Kaufman and Sharp,1982 Mol. Biol. 159:601-621), NSO myeloma cells (where a GS expressionsystem as described in WO 87/04462, WO 89/01036, and EP 338,841 may beused), COS cells, SP2 cells, HeLa cells, baby hamster kidney cells,YB2/0 rat myeloma cells, human embryonic kidney cells, human embryonicretina cells, and others comprising the nucleic acid or antagonistsdisclosed herein form additional embodiments of the present invention;the preceding cited disclosures of which are incorporated herein byreference. Specific embodiments of the present invention comprisingnucleic acid encoding disclosed PCSK9-specific antagonists include, butare not limited to, E. coli; see, e.g., Pluckthun, 1991 Bio/Technology9:545-551, or yeast, such as Pichia, and recombinant derivatives thereof(see, e.g., Li et al., 2006 Nat. Biotechnol. 24:210-215); the precedingdisclosures of which are incorporated herein by reference. Specificembodiments of the present invention relate to eukaryotic cellscomprising nucleic acid encoding the disclosed PCSK9-specificantagonists, see, Chadd & Chamow, 2001 Current Opinion in Biotechnology12:188-194, Andersen & Krummen, 2002 Current Opinion in Biotechnology13:117, Larrick & Thomas, 2001 Current Opinion in Biotechnology12:411-418; the disclosures of which are incorporated herein byreference. Specific embodiments of the present invention relate tomammalian cells comprising nucleic acid encoding the disclosedPCSK9-specific antagonists which are able to produce PCSK9-specificantagonists with proper post translational modifications. Posttranslational modifications include, but are by no means limited to,disulfide bond formation and glycosylation. Another type of posttranslational modification is signal peptide cleavage. Preferredembodiments herein have the appropriate glycosylation; see, e., Yoo etal., 2002 J. Immunol. Methods 261:1-20; the disclosure of which isincorporated herein by reference. Naturally occurring antibodies containat least one N-linked carbohydrate attached to a heavy chain. Id.Different types of mammalian host cells can be used to provide forefficient post-translational modifications. Examples of such host cellsinclude Chinese Hamster Ovary (CHO), HeLa, C6, PC12, and myeloma cells;see, Yoo et al., 2002 J. Immunol. Methods 261: 1-20, and Persic et al.,1997 Gene 187:9-18; the disclosures of which are incorporated herein byreference.

In another aspect, the present invention provides isolated cell(s)comprising a polypeptide of the present invention.

In another aspect, the present invention provides a method of making aPCSK9-specific antagonist of the present invention, which comprisesincubating a cell comprising nucleic acid encoding the PCSK9-specificantagonist, or a heavy and/or light chain or a fragment thereof (e.g.,VH and/or VL, or one or more of the disclosed heavy and/or light chainvariable region CDRs) of a desired PCSK9-specific antagonist (dictatedby the desired antagonist) with specificity for human and/or murinePCSK9 under conditions that allow the expression of the PCSK9-specificantagonist, or the expression and assembly of said heavy and/or lightchains or fragment into a PCSK9-specific antagonist, and isolating saidPCSK9-specific antagonist from the cell. One example by which togenerate particular desired heavy and/or light chain sequence orfragment is to first amplify (and modify) the germline heavy and/orlight chain variable sequences or fragment using PCR. Germline sequencefor human heavy and/or light variable regions are readily available tothe skilled artisan, see, e.g., the “Vbase” human germline sequencedatabase, and Kabat, E. A. et al., 1991 Sequences of Proteins ofImmunological Interest, Fifth Edition, U.S. Department of Health andHuman Services, NIH Publication No. 91-3242; Tomlinson, I. M. et al.,1992 “The Repertoire of Human Germline VH Sequences Reveals about FiftyGroups of VH Segments with Different Hypervariable Loops” J. Mol. Biol.227:776-798; and Cox, J. P. L. et al., 1994 “A Directory of HumanGerm-line Vκ Segments Reveals a Strong Bias in their Usage” Eur. J.Immunol. 24:827-836; the disclosures of which are incorporated herein byreference. Mutagenesis of germline sequences may be carried out usingstandard methods, e.g., PCR-mediated mutagenesis where the mutations areincorporated into PCR primers, or site-directed mutagenesis. Iffull-length antibodies are desired, sequence is available for the humanheavy chain constant region genes; see, e.g., Kabat. E. A. et al., 1991Sequences of Proteins of Immunological Interest, Fifth Edition, U.S.Department of Health and Human Services, NIH Publication No. 91-3242.Fragments containing these regions may be obtained, for example, bystandard PCR amplification. Alternatively, the skilled artisan can availhim/herself of vectors already encoding heavy and/or light chainconstant regions.

Available techniques exist to recombinantly produce other antibodymolecules which retain the specificity of an original antibody. Aspecific example of this is where DNA encoding the immunoglobulinvariable region or the CDRs is introduced into the constant regions, orconstant regions and framework regions, or simply the framework regions,of another antibody molecule; see, e.g., EP-184,187, GB 2188638, andEP-239400; the disclosures of which are incorporated herein byreference. Cloning and expression of antibody molecules, includingchimeric antibodies, are described in the literature; see, e.g., EP0120694 and EP 0125023; the disclosures of which are incorporated hereinby reference.

Antibody molecules in accordance with the present invention may, in oneinstance, be raised and then screened for characteristics identifiedherein using known techniques. Basic techniques for the preparation ofmonoclonal antibodies are described in the literature, see, e.g., Kohlerand Milstein (1975, Nature 256:495-497); the disclosure of which isincorporated herein by reference. Fully human monoclonal antibodies canbe produced by available methods. These methods include, but are by nomeans limited to, the use of genetically engineered mouse strains whichpossess an immune system whereby the mouse antibody genes have beeninactivated and in turn replaced with a repertoire of functional humanantibody genes, while leaving other components of the mouse immunesystem unchanged. Such genetically engineered mice allow for the naturalin vivo immune response and affinity maturation process which results inhigh affinity, full human monoclonal antibodies. This technology is wellknown in the art and is fully detailed in various publications,including but not limited to U.S. Pat. Nos. 5,545,806; 5,569,825;5,625,126; 5,633,425; 5,789,650; 5,877,397; 5,661,016; 5,814,318;5,874,299; 5,770,249 (assigned to GenPharm International and availablethrough Medarex, under the umbrella of the “UltraMab Human AntibodyDevelopment System”); as well as U.S. Pat. Nos. 5,939,598; 6,075,181;6,114,598; 6,150,584 and related family members (assigned to Abgenix,disclosing their XenoMouse® technology); the disclosures of which areincorporated herein by reference. See also reviews from Kellerman andGreen, 2002 Curr. Opinion in Biotechnology 13:593-597, and Kontermann &Stefan, 2001 Antibody Engineering, Springer Laboratory Manuals; thedisclosures of which are incorporated herein by reference.

Alternatively, a library of PCSK9-specific antagonists in accordancewith the present invention may be brought into contact with PCSK9, andones able to demonstrate specific binding selected. Functional studiescan then be carried out to ensure proper functionality, e.g., inhibitionof PCSK9-dependent inhibition of cellular LDL uptake. There are varioustechniques available to the skilled artisan for the selection ofprotein-specific molecules from libraries using enrichment technologiesincluding, but not limited to, phage display (e.g., see technology fromCambridge Antibody Technology (“CAT”) disclosed in U.S. Pat. Nos.5,565,332; 5,733,743; 5,871,907; 5,872,215; 5,885,793; 5,962,255;6,140,471; 6,225,447; 6,291,650; 6,492,160; 6,521,404; 6,544,731;6,555,313; 6,582,915; 6,593,081, as well as other U.S. family membersand/or applications which rely on priority filing GB 9206318, filed May24, 1992; see also Vaughn et al., 1996, Nature Biotechnology14:309-314), ribosome display (see, e.g., Hanes and Pluckthün, 1997Proc. Natl. Acad. Sci. 94:4937-4942), bacterial display (see, e.g.,Georgiou, et al., 1997 Nature Biotechnology 15:29-34) and/or yeastdisplay (see, e.g., Kieke, et al., 1997 Protein Engineering10:1303-1310); the preceding disclosures of which are incorporatedherein by reference. A library, for example, can be displayed on thesurface of bacteriophage particles, with nucleic acid encoding thePCSK9-specific antagonist or fragment thereof expressed and displayed onits surface. Nucleic acid may then be isolated from bacteriophageparticles exhibiting the desired level of activity and the nucleic acidused in the development of desired antagonist. Phage display has beenthoroughly described in the literature; see, e.g., Kontermann & Stefan,supra, and International Application Number WO 92/01047; the disclosuresof which are incorporated herein by reference. Specifically with regardto antibody molecules, individual heavy or light chain clones inaccordance with the present invention may also be used to screen forcomplementary heavy or light chains, respectively, capable ofinteraction therewith to form a molecule of the combined heavy and lightchains; see, e.g., International Application Number WO 92/01047. Anymethod of panning which is available to the skilled artisan may be usedto identify PCSK9-specific antagonists. Another specific method foraccomplishing this is to pan against the target antigen in solution,e.g. biotinylated, soluble PCSK9, and then capture the PCSK9-specificantagonist-phage complexes on streptavidin-coated magnetic beads, whichare then washed to remove nonspecifically-bound phage. The capturedphage can then be recovered from the beads in the same way they would berecovered from the surface of a plate, (e.g. DTT) as described herein.

PCSK9-specific antagonists may be purified by techniques available toone of skill in the art. Titers of the relevant antagonist preparation,ascites, hybridoma culture fluids, or relevant sample may be determinedby various serological or immunological assays which include, but arenot limited to, precipitation, passive agglutination, enzyme-linkedimmunosorbent antibody (“ELISA”) techniques and radioimmunoassay (“RIA”)techniques.

The present invention relates in part to methods employingPCSK9-specific antagonists described herein for antagonizing PCSK9function; said methods of which are further described below. Use of theterm “antagonizing” throughout the present application refers to the actof opposing, inhibiting, counteracting, neutralizing or curtailing oneor more functions of PCSK9. Inhibition or antagonism of one or more ofPCSK9-associated functional properties can be readily determinedaccording to methodologies known to the art (see, e.g., Barak & Webb,1981 J. Cell Biol. 90:595-604; Stephan & Yurachek, 1993 J Lipid Res.34:325330; and McNamara et al., 2006 Clinica Chimica Acta 369:158-167)as well as those described herein. Inhibition or antagonism willeffectuate a decrease in PCSK9 activity relative to that seen in theabsence of the antagonist or, for example, that seen when a controlantagonist of irrelevant specificity is present. Preferably, aPCSK9-specific antagonist in accordance with the present inventionantagonizes PCSK9 functioning to the point that there is a decrease ofat least 10%, of the measured parameter including but not limited to theactivities disclosed herein, and more preferably, a decrease of at least20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% and 95% of the measuredparameter. Such inhibition/antagonism of PCSK9 functioning isparticularly effective in those instances where PCSK9 functioning iscontributing at least in part to a particular phenotype, disease,disorder or condition which is negatively impacting the subject.

In one aspect, the present invention provides a method for antagonizingthe activity of PCSK9, which comprises contacting a cell, population ofcells or tissue sample capable of being affected by PCSK9 (i.e., whichexpresses and/or comprises LDL receptors) with a PCSK9-specificantagonist disclosed herein under conditions that allow said antagonistto bind to PCSK9 when present and inhibit PCSK9's inhibition of cellularLDL uptake. Specific embodiments of the present invention include suchmethods wherein the cell is a human cell. Additional embodiments of thepresent invention include such methods wherein the cell is a murinecell.

In another aspect, the present invention provides a method forantagonizing the activity of PCSK9 in a subject, which comprisesadministering to the subject a therapeutically effective amount of aPCSK9-specific antagonist of the present invention. In specificembodiments, the methods for antagonizing PCSK9 function are for thetreatment of a PCSK9-associated disease, disorder or condition or,alternatively, a disease, disorder or condition that could benefit fromthe effects of a PCSK9 antagonist. The medicament would be useful in asubject(s) exhibiting a condition associated with PCSK9 activity, or acondition where the functioning of PCSK9 is contraindicated for aparticular subject. In select embodiments, the condition may behypercholesterolemia, coronary heart disease, metabolic syndrome, acutecoronary syndrome or related conditions.

The present invention, thus, contemplates the use of PCSK9-specificantagonists described herein in various methods of treatment whereantagonizing PCSK9 function is desirable. The method of treatment can beprophylactic or therapeutic in nature. In specific embodiments, thepresent invention relates to a method of treatment for a conditionassociated with/attributed to PCSK9 activity, or a condition where thefunctioning of PCSK9 is contraindicated for a particular subject, whichcomprises administering to the subject a therapeutically effectiveamount of a PCSK9-specific antagonist of the present invention. Inselect embodiments, the condition may be hypercholesterolemia, coronaryheart disease, metabolic syndrome, acute coronary syndrome or relatedconditions.

Methods of treatment in accordance with the present invention compriseadministering to an individual a therapeutically (or prophylactically)effective amount of a PCSK9-specific antagonist of the presentinvention. Use of the terms “therapeutically effective” or“prophylactically effective” in reference to an amount refers to theamount necessary at the intended dosage to achieve the desiredtherapeutic/prophylactic effect for the period of time desired. Thedesired effect may be, for example, amelioration of at least one symptomassociated with the treated condition. These amounts will vary, as theskilled artisan will appreciate, according to various factors, includingbut not limited to the disease state, age, sex and weight of theindividual, and the ability of the PCSK9-specific antagonist to elicitthe desired effect in the individual. The response may be documented byin vitro assay, in vivo non-human animal studies, and/or furthersupported from clinical trials.

The PCSK9-specific antagonist may be administered as a pharmaceuticalcomposition. The present invention, thus, provides a pharmaceuticallyacceptable composition comprising a PCSK9-specific antagonist of theinvention and a pharmaceutically acceptable carrier including but notlimited to an excipient, diluent, stabilizer, buffer, or alternativedesigned to facilitate administration of the antagonist in the desiredformat and amount to the treated individual.

The pharmaceutical composition may be formulated by any number ofstrategies known in the art, see, e.g., McGoff and Scher, 2000 SolutionFormulation of Proteins/Peptides: In—McNally, E. J., ed. ProteinFormulation and Delivery. New York, N.Y.: Marcel Dekker; pp. 139-158;Akers & Defilippis, 2000, Peptides and Proteins as Parenteral Solutions.In—Pharmaceutical Formulation Development of Peptides and Proteins.Philadelphia, Pa.: Taylor and Francis; pp. 145-177; Akers et al., 2002,Pharm. Biotechnol. 14:47-127. A pharmaceutically acceptable compositionsuitable for patient administration will contain an effective amount ofthe PCSK9-specific antagonist in a formulation which both retainsbiological activity while also promoting maximal stability duringstorage within an acceptable temperature range.

The antagonist-based pharmaceutically acceptable composition may, inparticular embodiments, be in liquid or solid form, or in the form ofgas particles or aerosolized particles. Any technique for production ofliquid or solid formulations may be utilized. Such techniques are wellwithin the realm of the abilities of the skilled artisan. Solidformulations may be produced by any available method including, but notlimited to, lyophilization, spray drying, or drying by supercriticalfluid technology. Solid formulations for oral administration may be inany form rendering the antagonist accessible to the patient in theprescribed amount and within the prescribed period of time. The oralformulation can take the form of a number of solid formulationsincluding, but not limited to, a tablet, capsule, or powder. Solidformulations may alternatively be lyophilized and brought into solutionprior to administration for either single or multiple dosing accordingto methods well known to the skilled artisan. Antagonist compositionsshould generally be formulated within a biologically relevant pH rangeand may be buffered to maintain a proper pH range during storage. Bothliquid and solid formulations generally require storage at lowertemperatures (e.g., 2-8° C.) in order to retain stability for longerperiods. Formulated antagonist compositions, especially liquidformulations, may contain a bacteriostat to prevent or minimizeproteolysis during storage, including but not limited to effectiveconcentrations (e.g., ≦1% w/v) of benzyl alcohol, phenol, m-cresol,chlorobutanol, methylparaben, and/or propylparaben. A bacteriostat maybe contraindicated for some patients. Therefore, a lyophilizedformulation may be reconstituted in a solution either containing or notcontaining such a component. Additional components may be added toeither a buffered liquid or solid antagonist formulation, including butnot limited to sugars as a cryoprotectant (including but not limited topolyhydroxy hydrocarbons such as sorbitol, mannitol, glycerol, anddulcitol and/or disaccharides such as sucrose, lactose, maltose, ortrehalose) and, in some instances, a relevant salt (including but notlimited to NaCl, KCl, or LiCl). Such antagonist formulations, especiallyliquid formulations slated for long term storage, will rely on a usefulrange of total osmolarity to both promote long term stability attemperatures of, for example, 2-8° C. or higher, while also making theformulation useful for parenteral injection. As appropriate,preservatives, stabilizers, buffers, antioxidants and/or other additivesmay be included. The formulations may contain a divalent cation(including but not limited to MgCl2, CaCl2, and MnCl2); and/or anon-ionic surfactant (including but not limited to Polysorbate-80 (Tween80™), Polysorbate-60 (Tween 60™), Polysorbate-40 (Tween 40™), andPolysorbate-20 (Tween 20™), polyoxyethylene alkyl ethers, including butnot limited to Brij 58™, Brij35™, as well as others such as TritonX-100™, Triton X-114™, NP40™, Span 85 and the Pluronic series ofnon-ionic surfactants (e.g., Pluronic 121)). Any combination of suchcomponents form specific embodiments of the present invention.

Pharmaceutical compositions in liquid format may include a liquidcarrier, e.g., water, petroleum, animal oil, vegetable oil, mineral oil,or synthetic oil. The liquid format may also include physiologicalsaline solution, dextrose or other saccharide solution or glycols, suchas ethylene glycol, propylene glycol or polyethylene glycol.

Preferably, the pharmaceutical composition may be in the form of aparenterally acceptable aqueous solution that is pyrogen-free withsuitable pH, tonicity, and stability. Pharmaceutical compositions may beformulated for administration after dilution in isotonic vehicles, forexample, Sodium Chloride Injection, Ringer's Injection, or LactatedRinger's Injection.

One aspect of the present invention is a pharmaceutical compositionwhich comprises: (i) about 50 to about 200 mg/mL of protein includingbut not limited to the PCSK9-specific antagonists described herein; (ii)a polyhydroxy hydrocarbon (including but not limited to sorbitol,mannitol, glycerol and dulcitol) and/or a disaccharide (including butnot limited to sucrose, lactose, maltose and trehalose); the total ofsaid polyhydroxy hydrocarbon and/or disaccharide being about 1% to about6% weight per volume (“w/v”) of the formulation; (iii) about 5 mM toabout 200 mM of histidine, imidazole, phosphate or acetic acid whichserves as a buffering agent to prevent pH drift over the shelf life ofthe pharmaceutical composition and as a tonicity modifier; (iv) about 5mM to about 200 mM of arginine, proline, phenylalanine, alanine,glycine, lysine, glutamic acid, aspartic acid or methionine tocounteract aggregation; (v) about 0.01 M to about 0.1 M of hydrochloricacid (“HCl”) in an amount sufficient to achieve a pH in the range ofabout 5.5 to about 7.5; and (vi) a liquid carrier including but notlimited to sterile water, petroleum, animal oil, vegetable oil, mineraloil, synthetic oil, physiological saline solution, dextrose or othersaccharide solution or glycols, such as ethylene glycol, propyleneglycol or polyethylene glycol; wherein said pharmaceutical compositionhas a pH in the range of about 5.5 to about 7.5; and wherein saidpharmaceutical composition optionally comprises about 0.01% to about 1%w/v of the formulation of a non-ionic surfactant (including but notlimited to Polysorbate-80 (Tween 80™), Polysorbate-60 (Tween 60™),Polysorbate-40 (Tween 40™), and Polysorbate-20 (Tween 20™),polyoxyethylene alkyl ethers, including but not limited to Brij 58™,Brij35™, as well as others such as Triton X-100™, Triton X-114™, NP40™,Span 85 and the Pluronic series of non-ionic surfactants (e.g., Pluronic121)).

HCl may be added as free acid, Histidine-HCl or Arginine-HCl. Wheresupplied as Histidine-HCl or Arginine-HCl, the total amounts ofHistidine or Arginine in the HCl form should be that specified above.Accordingly, some or all of the HCl depending on the amounts ofHistidine and/or Arginine may be supplied as Histidine-HCl and/orArginine-HCl; as appropriate. Use of the term “about” with respect toamounts disclosed in the specification means within 10% of the specifiednumbers provided. A range provided as, for example” in “about 50 toabout 200” expressly includes as distinct embodiments each number withinsaid range. As such in the above example, embodiments including but notlimited to those having 50, 100, 125, 150 and 200 form specificembodiments herein. Pharmaceutical compositions as disclosed herein havegeneral applicability despite the mode of administration. In specificembodiments, the disclosed pharmaceutical compositions are useful forsubcutaneous administration as a liquid or upon reconstitution of alyophilized form. Proteins that can be employed in the disclosedformulations include any polymeric protein or polypeptide characterizedas comprising covalently linked amino acid residues delivered forpurposes of effecting a therapeutic benefit. Proteins of use in thepresent compositions include but are not limited to any antibodymolecules as defined herein or any non-antibody or non-immunoglobulinproteins, peptides, pegylated proteins and fusion proteins.

Specific aspects of the present invention relate to the above disclosedpharmaceutical compositions which comprise: (i) about 50 to about 200mg/mL of protein including but not limited to the PCSK9-specificantagonists described herein; (ii) about 1% to about 6% (in particularembodiments from about 2% to about 6%) w/v mannitol, trehalose orsucrose; (iii) about 10 mM to about 100 mM of histidine; (iv) about 25mM to about 100 mM of arginine or proline; (v) about 0.02 M to about0.05M of hydrochloric acid (“HCl”) in an amount sufficient to achieve apH in the range of about 5.8 to about 7; and (vi) a liquid carrierincluding but not limited to sterile water, petroleum, animal oil,vegetable oil, mineral oil, synthetic oil, physiological salinesolution, dextrose or other saccharide solution or glycols, such asethylene glycol, propylene glycol or polyethylene glycol; wherein saidpharmaceutical composition has a pH in the range of about 5.8 to about7; and wherein said pharmaceutical composition optionally comprisingabout 0.01% to about 1% w/v of the formulation of a non-ionic surfactant(including but not limited to Polysorbate-80 (Tween 80™), Polysorbate-60(Tween 60™), Polysorbate-40 (Tween 40™), and Polysorbate-20 (Tween 20™),polyoxyethylene alkyl ethers, including but not limited to Brij 58™,Brij35™, as well as others such as Triton X-100™, Triton X-114™, NP40™,Span 85 and the Pluronic series of non-ionic surfactants (e.g., Pluronic121)).

Specific embodiments provide pharmaceutical compositions which comprise:(i) 50 to 200 mg/mL of protein including but not limited to thePCSK9-specific antagonists described herein; (ii) about 1% to about 6%(in particular embodiments from about 2% to about 6%) w/v mannitol,trehalose or sucrose; (iii) about 10 mM to about 150 mM of histidine;(iv) about 10 mM to about 150 mM of arginine or proline; (v) about 0.03M to about 0.05 M of hydrochloric acid (“HCl”) in an amount sufficientto achieve a pH in the range of about 5.8 to about 6.5; and (vi) aliquid carrier including but not limited to sterile water, petroleum,animal oil, vegetable oil, mineral oil, synthetic oil, physiologicalsaline solution, dextrose or other saccharide solution or glycols, suchas ethylene glycol, propylene glycol or polyethylene glycol; whereinsaid pharmaceutical composition has a pH in the range of about 5.8 toabout 6.5; and wherein said pharmaceutical composition optionallycomprising about 0.01% to about 1% w/v of Polysorbate-80 (Tween 80™) orPolysorbate-20 (Tween 20™).

Specific embodiments herein provide pharmaceutical compositions whichcomprise: (i) 50 to 200 mg/mL of protein including but not limited tothe PCSK9-specific antagonists described herein; (ii) about 1% to about6% (in particular embodiments from about 2% to about 6%) w/v sucrose;(iii) about 25 mM to about 100 mM of histidine; (iv) about 25 mM toabout 100 mM of arginine; (v) about 0.040 M to about 0.045 M ofhydrochloric acid (“HCl”) in an amount sufficient to achieve a pH ofabout 6; and (vi) sterile water; wherein said pharmaceutical compositionhas a pH of about 6; and wherein said pharmaceutical compositionoptionally comprising about 0.01% to about 1% w/v of Polysorbate-80(Tween 80™) or Polysorbate-20 (Tween 20™). In specific embodimentsthereof, the levels of histidine and arginine are within 25 mM of eachother and, in other embodiments are the same.

Specific embodiments herein provide pharmaceutical compositions whichcomprise (i) 50 to 200 mg/mL of protein including but not limited to thePCSK9-specific antagonists described herein; (ii) sucrose, histidine andarginine in one of the following amounts: (a) about 1% w/v sucrose,about 10 mM histidine and about 25 mM arginine; (b) about 2% w/vsucrose, about 25 mM histidine and about 25 mM arginine; (c) about 3%w/v sucrose, about 50 mM histidine and about 50 mM arginine; or (d)about 6% w/v sucrose, about 100 mM histidine and about 100 mM arginine;(iii) about 0.04 mol or, alternatively, about 1.46 g of HCl; and (iv)sterile water; wherein said pharmaceutical composition has a pH of about6; and wherein said pharmaceutical composition optionally comprisingabout 0.01% to about 1% w/v of Polysorbate-80 (Tween 80™) orPolysorbate-20 (Tween 20™). Specific embodiments herein are wherein theamounts of sucrose, histidine and arginine in (ii) above are thatdescribed in (c) or (d). Specific embodiments employing pharmaceuticalformulations as described above wherein the amounts of sucrose,histidine and arginine are that specified in (ii) (c) were found toprovide an osmolality similar to the physiological value of 300 mOsm andprovided stability in both the liquid and lyophilized form.

Specific embodiments herein provide pharmaceutical compositions asdescribed which comprise 50 to 200 mg/ml of any one of the variousPCSK9-specific antagonists described herein. For purposes ofexemplification of one distinct embodiment thereof, and not to beconstrued as a limitation, is the following: a pharmaceuticalformulation as described above which comprises: a PCSK9-specificantagonist which comprises: (a) a light chain comprising SEQ ID NO: 26;and (b) a heavy chain comprising SEQ ID NO: 25; wherein saidPCSK9-specific antagonist is an antibody molecule that antagonizesPCSK9's inhibition of cellular LDL uptake.

Particular embodiments herein are pharmaceutical compositions accordingto the above description which are lyophilized and reconstituted. Inspecific embodiments, said protein concentration in said lyophilized andreconstituted solution is up to 2-fold higher than in thepre-lyophilized composition. In specific embodiments, the protein orPCSK9-specific antagonist concentration in the lyophilized and/orreconstituted pharmaceutical composition is in the range of about 50mg/mL to about 300 mg/mL. Diluents useful for reconstituting thelyophilized pharmaceutical compositions include but are not limited tosterile water, bacteriostatic water for injection (“BWFI”),phosphate-buffered saline, a sterile saline solution, physiologicalsaline solution, Ringer's solution or dextrose solution and may inspecific embodiments contain 0.01-1% (w/v) of Polysorbate-80 (Tween 80™)or Polysorbate-20 (Tween 20™). In specific embodiments, lyophilizedpowder can be reconstituted with 1/60.2× original volume (or 0.167 mL)up to 1× (1 mL).

Exemplary embodiments of the present invention are pharmaceuticalcompositions as described herein which are stable. Other embodiments ofthe present invention are pharmaceutical compositions as describedherein which are stable to lyophilization and reconstitution. Variousmethods are available to the skilled artisan to prepare lyophilizedcompositions; see, e.g., Martin & Mo, 2007 “Stability Considerations forLyophilized Biologics” Amer. Pharm. Rev. “Stable” as used herein refersto the property of the protein or PCSK9-specific antagonist to retainits physical or chemical stability, conformational integrity, or itsability to exhibit less denaturation, protein clipping, aggregation,fragmentation, acidic variant formation or loss of biological activitycompared with a control sample at a temperature in the range of 4-37° C.for at least about 30 days. Other embodiments remain stable for up to 3months, 6 months, 12 months, 2 years or longer periods at the abovetemperatures. In specific embodiments the formulation exhibits nosignificant changes at 2-8° C. for at least 6 months, and preferably 12months, 2 years or longer, in order of preference. Specific embodimentsexperience less than 10% or, in particular embodiments, less than 5% ofdenaturation, protein clipping, aggregation, fragmentation, acidicvariant formation or loss of biological activity compared with a controlsample at a temperature in the range of 25-45° C. (or alternatively 2-8°C.) for at least about 30 days, 3 months, 6 months, 12 months, 2 yearsor longer. Stability of the formulations can be tested via several meansknown to the skilled artisan including, but not limited to SizeExclusion Chromatography (SEC-HPLC) to measure aggregation andfragmentation, Dynamic Light Scattering (DLS) to measure particle sizeof concentrated samples, capillary SDS-PAGE to measure fragmentation andcapillary iso-electric focusing (cIEF) or cation exchange chromatography(“CEX”) to measure acidic variants formation. Techniques suitable forthe analysis of protein stability are well understood by those of skillin the art: see review in Peptide and Protein Drug Delivery, 247-301,Vincent Lee Ed., Marcel Dekker, Inc., New York, N.Y., Pubs. (1991) andJones, 1993 Adv. Drug Delivery Rev. 10:29-90.

Pharmaceutical compositions as described herein should be sterile. Thereare various techniques available to the skilled artisan to accomplishthis including, but not limited to, filtration through sterilefiltration membranes. In specific embodiments, employing lyophilized andreconstituted compositions, this may be done prior to or followinglyophilization and reconstitution.

Dosing of antagonist therapeutics is well within the realm of theskilled artisan, see, e.g., Lederman et al., 1991 Int. J. Cancer47:659-664; Bagshawe et al., 1991 Antibody, Immunoconjugates andRadiopharmaceuticals 4:915-922, and will vary based on a number offactors including but not limited to the particular PCSK9-specificantagonist utilized, the patient being treated, the condition of thepatient, the area being treated, the route of administration, and thetreatment desired. A physician or veterinarian of ordinary skill canreadily determine and prescribe the effective therapeutic amount of theantagonist. Dosage ranges may be from about 0.01 to 100 mg/kg, and moreusually 0.05 to 25 mg/kg, of the host body weight. For example, dosagescan be 0.3 mg/kg body weight, 1 mg/kg body weight, 3 mg/kg body weight,5 mg/kg body weight or 10 mg/kg body weight or within the range of 1-10mg/kg. For purposes of illustration, and not limitation, in specificembodiments, a dose of 5 mg to 2.0 g may be utilized to deliver theantagonist systemically. In specific embodiments, the concentration ofthe dose provided will be in the range of about 8 mg/mL to about 200mg/mL. In other embodiments, a dose contemplated for use in the presentinvention is from about 50 mg/mL to about 150 mg/mL. In specificembodiments, the dose will be from about 0.1 mL to about 1.5 mL and inspecific embodiments is 1 mL. Optimal precision in achievingconcentrations of antagonist within a range that yields efficacy withouttoxicity requires a regimen based on the kinetics of the drug'savailability to the target site(s). This involves a consideration of thedistribution, equilibrium, and elimination of the PCSK9-specificantagonist. Antagonists described herein may be used alone atappropriate dosages. Alternatively, co-administration or sequentialadministration of other agents may be desirable. It will be possible topresent a therapeutic dosing regime for the PCSK9-specific antagonistsof the present invention in conjunction with alternative treatmentregimes. For example, PCSK9-specific antagonists may be used incombination or in conjunction with other drugs (therapeutic and/orprophylactic), including but not limited to cholesterol-lowering drugs,for example, cholesterol absorption inhibitors (e.g., Zetia®) andcholesterol synthesis inhibitors (e.g., Zocor® and Vytorin®). Thepresent invention contemplates such combinations and they form animportant embodiment hereof. Accordingly, the present invention relatesto methods of treatment as described above where the PCSK9-specificantagonist is administered/delivered simultaneously with, following orprior to another drug or drugs (therapeutic and/or prophylactic),including but not limited to cholesterol-lowering drugs, cholesterolabsorption inhibitors and cholesterol absorption inhibitors.

Individuals (subjects) capable of treatment as described herein includeprimates, human and non-human, and include any non-human mammal orvertebrate of commercial or domestic veterinary importance.

The PCSK9-specific antagonist may be administered to an individual byany route of administration appreciated in the art, including but notlimited to oral administration, administration by injection (specificembodiments of which include intravenous, subcutaneous, intraperitonealor intramuscular injection), or administration by inhalation,intranasal, or topical administration, either alone or in combinationwith other agents designed to assist in the treatment of the individual.The PCSK9-specific antagonist may also be administered by injectiondevices, injector pens, needleless devices; and subcutaneous patchdelivery systems. The route of administration should be determined basedon a number of considerations appreciated by the skilled artisanincluding, but not limited to, the desired physiochemicalcharacteristics of the treatment. Treatment may be provided on a daily,weekly, biweekly, or monthly basis, or any other regimen that deliversthe appropriate amount of PCSK9-specific antagonist to the individual atthe prescribed times such that the desired treatment is effected andmaintained. The formulations may be administered in a single dose or inmore than one dose at separate times.

Also contemplated are methods of using the disclosed antagonists in themanufacture of a medicament for treatment of a PCSK9-associated disease,disorder or condition or, alternatively, a disease, disorder orcondition that could benefit from the effects of a PCSK9 antagonist. Themedicament would be useful in a subject(s) exhibiting a conditionassociated with PCSK9 activity, or a condition where the functioning ofPCSK9 is contraindicated fr a particular subject. In select embodiments,the condition may be hypercholesterolemia, coronary heart disease,metabolic syndrome, acute coronary syndrome or related conditions.

PCSK9-specific antagonists disclosed herein may also be used as a methodof diagnosis of PCSK9. In select embodiments, the present inventionencompasses methods of identifying or quantifying the level of PCSK9present in a sample (including but not limited to a biological sample,e.g., serum or blood) which comprises contacting the sample with aPCSK9-specific antagonist described herein and detecting or quantifying,respectively, binding to PCSK9. The PCSK9-specific antagonist may beused in various assay formats known to the skilled artisan and may formpart of a kit (the general features of a kit of which are furtherdescribed below).

The present invention further provides for the administration ofdisclosed anti-PCSK9 antagonists for purposes of gene therapy. Throughsuch methods, cells of a subject are transformed with nucleic acidencoding a PCSK9-specific antagonist of the invention. Subjectscomprising the nucleic acids then produce the PCSK9-specific antagonistsendogenously. Previously, Alvarez, et al, Clinical Cancer Research6:3081-3087, 2000, introduced single-chain anti-ErbB2 antibodies tosubjects using a gene therapy approach. The methods disclosed byAlvarez, et al, supra, may be easily adapted for the introduction ofnucleic acids encoding an anti-PCSK9 antibody of the invention to asubject.

Nucleic acids encoding any PCSK9-specific antagonist may be introducedto a subject.

The nucleic acids may be introduced to the cells of a subject by anymeans known in the art. In preferred embodiments, the nucleic acids areintroduced as part of a viral vector. Examples of preferred viruses fromwhich the vectors may be derived include lentiviruses, herpes viruses,adenoviruses, adeno-associated viruses, vaccinia virus, baculovirus,alphavirus, influenza virus, and other recombinant viruses withdesirable cellular tropism.

Various companies produce viral vectors commercially, including, but byno means limited to, Avigen, Inc. (Alameda, Calif.; AAV vectors), CellGenesys (Foster City, Calif.; retroviral, adenoviral, AAV vectors, andlentiviral vectors), Clontech (retroviral and baculoviral vectors),Genovo, Inc. (Sharon Hill, Pa.; adenoviral and AAV vectors), Genvec(adenoviral vectors), IntroGene (Leiden, Netherlands; adenoviralvectors), Molecular Medicine (retroviral, adenoviral, AAV, and herpesviral vectors), Norgen (adenoviral vectors), Oxford BioMedica (Oxford,United Kingdom; lentiviral vectors), and Transgene (Strasbourg, France;adenoviral, vaccinia, retroviral, and lentiviral vectors).

Methods for constructing and using viral vectors are known in the art(see, e.g., Miller, et al, BioTechniques 7:980-990, 1992). Preferably,the viral vectors are replication defective, that is, they are unable toreplicate autonomously, and thus are not infectious, in the target cell.Preferably, the replication defective virus is a minimal virus, i.e., itretains only the sequences of its genome which are necessary forencapsidating the genome to produce viral particles. Defective viruses,which entirely or almost entirely lack viral genes, are preferred. Useof defective viral vectors allows for administration to cells in aspecific, localized area, without concern that the vector can infectother cells. Thus, a specific tissue can be specifically targeted.

Examples of vectors comprising attenuated or defective DNA virussequences include, but are not limited to, a defective herpes virusvector (Kanno et al, Cancer Gen. Ther. 6:147-154, 1999; Kaplitt et al,J. Neurosci. Meth. 71:125-132, 1997 and Kaplitt et al, J. Neuro One.19:137-147, 1994).

Adenoviruses are eukaryotic DNA viruses that can be modified toefficiently deliver a nucleic acid of the invention to a variety of celltypes. Attenuated adenovirus vectors, such as the vector described byStrafford-Perricaudet et al, J. Clin. Invest. 90:626-630, 1992 aredesirable in some instances. Various replication defective adenovirusand minimum adenovirus vectors have been described (PCT Publication Nos.WO94/26914, WO94/28938, WO94/28152, WO94/12649, WO95/02697 andWO96/22378). The replication defective recombinant adenovirusesaccording to the invention can be prepared by any technique known to aperson skilled in the art (Levrero et al, Gene 101:195, 1991; EP 185573;Graham, EMBO J. 3:2917, 1984; Graham et al, J. Gen. Virol. 36:59, 1977).

The adeno-associated viruses (AAV) are DNA viruses of relatively smallsize which can integrate, in a stable and site-specific manner, into thegenome of the cells which they infect. They are able to infect a widespectrum of cells without inducing any effects on cellular growth,morphology or differentiation, and they do not appear to be involved inhuman pathologies. The use of vectors derived from the AAVs fortransferring genes in vitro and in vivo has been described (see Daly, etal, Gene Ther. 8:1343-1346, 2001, Larson et al, Adv. Exp. Med. Bio.489:45-57, 2001; PCT Publication Nos. WO 91/18088 and WO 93/09239; U.S.Pat. Nos. 4,797,368 and 5,139,941 and EP 488528B1).

In another embodiment, the gene can be introduced in a retroviralvector, e.g., as described in U.S. Pat. Nos. 5,399,346, 4,650,764,4,980,289, and 5,124,263; Mann et al, Cell 33:153, 1983; Markowitz etal, J. Virol., 62:1120, 1988; EP 453242 and EP 178220. The retrovirusesare integrating viruses which infect dividing cells.

Lentiviral vectors can be used as agents for the direct delivery andsustained expression of nucleic acids encoding a PCSK9-specificantagonist of the invention in several tissue types, including brain,retina, muscle, liver and blood. The vectors can efficiently transducedividing and nondividing cells in these tissues, and maintain long-termexpression of the PCSK9-specific antagonist. For a review, see Zuffereyet al, J. Virol. 72:9873-80, 1998 and Kafri et al, Curr. Opin. Mol.Ther. 3:316-326, 2001. Lentiviral packaging cell lines are available andknown generally in the art. They facilitate the production of high-titerlentivirus vectors for gene therapy. An example is atetracycline-inducible VSV-G pseudotyped lentivirus packaging cell linewhich can generate virus particles at titers greater than 10⁶ IU/ml forat least 3 to 4 days; see Kafri et al, J. Virol. 73:576-584, 1999. Thevector produced by the inducible cell line can be concentrated as neededfor efficiently transducing nondividing cells in vitro and in vivo.

Sindbis virus is a member of the alphavirus genus and has been studiedextensively since its discovery in various parts of the world beginningin 1953. Gene transduction based on alphavirus, particularly Sindbisvirus, has been well-studied in vitro (see Straus et al, Microbiol.Rev., 58:491-562, 1994; Bredenbeek et al, J. Virol., 67:6439-6446, 1993;Ijima et al, Int. J. Cancer 80:110-118, 1999 and Sawai et al, Biochim.Biophyr. Res. Comm. 248:315-323, 1998. Many properties of alphavirusvectors make them a desirable alternative to other virus-derived vectorsystems being developed, including rapid engineering of expressionconstructs, production of high-titered stocks of infectious particles,infection of nondividing cells, and high levels of expression (Strausset al, 1994 supra). Use of Sindbis virus for gene therapy has beendescribed. (Wahlfors et al, Gene. Ther. 7:472-480, 2000 and Lundstrom,J. Recep. Sig. Transduct. Res. 19(1-4):673-686, 1999.

In another embodiment, a vector can be introduced to cells bylipofection or with other transfection facilitating agents (peptides,polymers, etc.). Synthetic cationic lipids can be used to prepareliposomes for in vivo and in vitro transfection of a gene encoding amarker (Feigner et al, Proc. Natl. Acad. Sci. USA 84:7413-7417, 1987 andWang et al, Proc. Natl. Acad. Sci. USA 84:7851-7855, 1987). Useful lipidcompounds and compositions for transfer of nucleic acids are describedin PCT Publication Nos. WO 95/18863 and WO 96/17823, and in U.S. Pat.No. 5,459,127.

It is also possible to introduce the vector in vivo as a naked DNAplasmid. Naked DNA vectors for gene therapy can be introduced intodesired host cells by methods known in the art, e.g., electroporation,microinjection, cell fusion, DEAE dextran, calcium phosphateprecipitation, use of a gene gun, or use of a DNA vector transporter(see, e.g., Wilson, et al, J. Biol. Chem. 267:963-967, 1992; Williams etal, Proc. Natl. Acad. Sci. USA 88:2726-2730, 1991). Other reagentscommonly used for transfection of plasmids include, but are by no meanslimited to, FuGene, Lipofectin, and Lipofectamine. Receptor-mediated DNAdelivery approaches can also be used (Wu et al, J. Biol. Chem.263:14621-14624, 1988). U.S. Pat. Nos. 5,580,859 and 5,589,466 disclosedelivery of exogenous DNA sequences, free of transfection facilitatingagents, in a mammal. Recently, a relatively low voltage, high efficiencyin vivo DNA transfer technique, termed electrotransfer, has beendescribed (Vilquin et al, Gene Ther. 8:1097, 2001; Payen et al, Exp.Hematol. 29:295-300, 2001; Mir, Bioelectrochemistry 53:1-10, 2001; PCTPublication Nos. WO 99/01157, WO 99/01158 and WO 99/01175).

Pharmaceutical compositions suitable for such gene therapy approachesand comprising nucleic acids encoding an anti-PCSK9 antagonist of thepresent invention are included within the scope of the presentinvention.

In another aspect, the present invention provides a method foridentifying, isolating, quantifying or antagonizing PCSK9 in a sample ofinterest using a PCSK9-specific antagonist of the present invention. ThePCSK9-specific antagonists may be utilized as research tools inimmunochemical assays, such as Western blots, ELISAs, radioimmunoassay,immunohistochemical assays, immunoprecipitations, or otherimmunochemical assays known in the art (see, e.g., ImmunologicalTechniques Laboratory Manual, ed. Goers, J. 1993, Academic Press) orvarious purification protocols. The antagonists may have a labelincorporated therein or affixed thereto to facilitate readyidentification or measurement of the activities associated therewith.One skilled in the art is readily familiar with the various types ofdetectable labels (e.g., enzymes, dyes, or other suitable moleculeswhich are either readily detectable or cause some activity/result thatis readily detectable) which are or may be useful in the aboveprotocols.

An additional aspect of the present invention are kits comprisingPCSK9-specific antagonists or pharmaceutical compositions disclosedherein and instructions for use. Kits typically but need not include alabel indicating the intended use of the contents of the kit. The termlabel includes any writing, or recorded material supplied on or with thekit, or which otherwise accompanies the kit. In specific embodimentswherein the pharmaceutical composition is provided lyophilized, the kitmay include sterile water or saline for reconstitution of theformulation into liquid form. In specific embodiments, the amount ofwater or saline is from about 0.1 ml to 1.0 ml.

The following examples are provided to illustrate the present inventionwithout limiting the same hereto:

EXAMPLE 1 Isolation of Recombinant Fab Display Phage 1B20

Recombinant Morphosys HuCAL Gold Fab phage display libraries (see, e.g.,Knappik et al., 2000 J. Mol. Biol. 296:57-86) were panned againstimmobilized recombinant human and murine PCSK9 through a process whichis briefly described as follows: PCSK9 protein was chemicallybiotinylated (Pierce, Cat. #21455) per manufacturer's instruction. TheMoprhosys phage Fab display libraries were pooled and pre-absorbed threetimes to blocked strepavidin coated beads (Dynal beads M280). With thegoal of isolating cross-reactive Fabs, human (h) and mouse (m) PCSK9were alternated as follows: rounds 1/2/3 utilized PCSK9 from h/m/h.

For each of the three rounds of panning, the preabsorbed phage librarywas incubated with preblocked biotinylated PCSK9 (150 nM for first roundand 100 nM for subsequent rounds) immobilized to strepavidin coatedDynal beads. The immobilized phage-PCSK9 complexes were washedsequentially with 5 quick washes with PBS/0.05% Tween™ 20 followed by 4quick washes with PBS and transferred in PBS to a fresh blocked tube.Bound phages were then eluted with 20 mM DTT. TG1 cells were infectedwith eluted phages. Pooled cultures of phagemid-bearing cells(chloramphenicol-resistant) were grown up and frozen stocks ofphagemid-bearing cultures were made. Phage were rescued from culture byco-infection with helper phage, and phage stocks for next round ofpanning were made.

After the third round of panning phagemid-infected cells were grownovernight and phagemid DNA was prepared.

XbaI-EcoRI inserts from Round 3 phagemid DNA were subcloned intoMorphosys Fab expression vector pMORPH_x9_MH to yield plasmidpMORPHx9_MH/PCSK9_(—)6_CX1_B20 (see, e.g., FIG. 1), and a library of Fabexpression clones was generated in E. coli TG1 F−. Transformants werespread on LB+chloramphenicol+glucose plates and grown overnight togenerate bacterial colonies. Individual transformant colonies werepicked and placed into wells of two 96-well plates for growth andscreening for Fab expression.

EXAMPLE 2 Elisa Screening of Bacterially Expressed Fabs

Cultures of individual transformants were IPTG-induced and grownovernight for Fab expression. Culture supernatants (candidate Fabs) wereincubated with purified V5-, His-tagged PCSK9 protein immobilized inwells of 96-well Nunc Maxisorp plates, washed with 0.1% Tween™ 20 in PBSusing a plate washer, incubated with HRP-coupled anti-Fab antibody, andwashed again with PBS/Tween™ 20. Bound HRP was detected by addition ofTMP substrate, and A450 values of wells were read with a plate reader.

Negative controls were included as follows:

Controls for nonspecific Fab binding on each plate were incubated withparallel expressed preparations of anti-EsB, an irrelevant Fab.

Growth medium only.

Positive controls for ELISA and Fab expression were included as follows:EsB antigen was bound to three wells of the plate and subsequentlyincubated with anti-EsB Fab. To control for Fabs reacting with the V5 orHis tags of the recombinant PCSK9 antigen, parallel ELISAs wereperformed using V5-, His-tagged secreted alkaline phosphatase protein(SEAP) expressed in the same cells as the original PCSK9 antigen andsimilarly purified. Putative PCSK9-reactive Fabs were identified asyielding >3× background values when incubated with PCSK9 antigen butnegative when incubated with SEAP. Clones scoring as PCSK9-reactive inthe first round of screening were consolidated onto a single plate,re-grown in triplicate, re-induced with IPTG, and re-assayed in parallelELISAs vs. PCSK9 and SEAP. Positive and negative controls were includedas described above. Clones scoring positive in at least 2 of 3replicates were carried forward into subsequent characterizations. Incases of known or suspected mixed preliminary clones, cultures werere-purified by streaking for single colonies on 2×YT plates withchloramphenicol, and liquid cultures from three or more separatecolonies were assayed again by ELISAs in triplicate as described above.

EXAMPLE 3 DNA Sequence Determination of PCSK9 Elisa-Positive Fab Clones

Bacterial cultures for DNA preps were made by inoculating 1.2 ml 2×YTliquid media with chloramphenicol from master glycerol stocks ofpositive Fabs, and growing overnight. DNA was prepared from cell pelletscentrifuged out of the overnight cultures using the Qiagen Turbo Minipreps performed on a BioRobot 9600. ABI Dye Terminator cycle sequencingwas performed on the DNA with Morphosys defined sequencing primers andrun on an ABI 3100 Genetic Analyzer, to obtain the DNA sequence of theFab clones. DNA sequences were compared to each other to determineunique clone sequences and to determine light and heavy chain subtypesof the Fab clones.

EXAMPLE 4 Expression and Purification of Fabs from unique PCSK9Elisa-Positive Clone

Fabs from ELISA-positive clone 1B20 and the EsB (negative control) Fabwere expressed by IPTG-induction in E. coli TG1F-cells. Cultures werelysed and the His-tagged Fabs were purified by immobilized metal ionaffinity chromatography (IMAC), and proteins were exchanged into 25 mMHEPES pH 7.3/150 mM NaCl by centrifugal diafiltration. Proteins wereanalyzed by electrophoresis on Caliper Lab-Chip 90 and by conventionalSDS-PAGE, and quantified by Bradford protein assay. Purified Fab proteinwas re-assayed by ELISA in serial dilutions to confirm activity ofpurified Fab. Positive and Negative controls were run as before.Purified Fab preparations were then analyzed as described below.

EXAMPLE 5 Conversion of 1B20 Fab to Full Length IgG

The DNA sequence encoding the 1B20 light kappa chain variable region wasamplified by polymerase chain reaction from plasmid templatepMORPHx9_MH/PCSK9_(—)6_CX1_B20, using forward primer5′-ACAGATGCCAGATGCGATATCGTGATGACCCAGA-3′ (SEQ ID NO: 31) and reverseprimer 5′-TGCAGCCACCGTACGTTTAATTTCAACTTTCGTACC-3′ (SEQ ID NO: 32). Theproduct of this amplification was cloned into plasmid pV 1JNSA-GS-FB-LCK that had been previously digested with FspI and BmtI,using the InFusion cloning system (Clontech). The resulting plasmid wasverified by DNA sequencing across the variable region. Endotoxin-freeplasmid preparations were made using the Qiagen Endo-Free plasmidmaxiprep kit.

The DNA sequence encoding the heavy gamma chain variable region ofpMORPHx9_MH/PCSK9_(—)6_CX1_B20 was amplified by polymerase chainreaction using forward primer 5′-ACAGGTGTCCACTCGCAGGTGCAATTGGTTCAGAGC-3′(SEQ ID NO: 33) and reverse primer5′-GCCCTTGGTGGATGCTGAGCTAACCGTCACCAGGGT-3′ (SEQ ID NO: 34), and theamplified product was cloned into plasmid pV1JNSA-BF-HCG2M4 that hadbeen previously digested with FspI and BmtI. The resulting plasmid wasverified by DNA sequencing across the variable region. Endotoxin-freeplasmid preparations were made using the Qiagen Endo-Free plasmidmaxiprep kit.

Full-length IgG was obtained by co-transfection of HEK293 cells with the1B20 light chain- and heavy-chain-encoding plasmids, following byProtein A purification of the expressed IgG.

EXAMPLE 6 Kinetic Evaluation of FAB:PCSK9 Interactions with SurfacePlasmon Resonance (“SPR”)

SPR measurements were performed using a Biacore™ (Pharmacia BiosensorAB, Uppsala, Sweden) 2000 system. Sensor chip CM5 and Amine Coupling Kitfor immobilization were from Biacore™.

Anti-Fab IgG (Human specific) (Sigma, catalog #15260) was covalentlycoupled to surfaces 1 and 2 of a Sensor Chip CM5 via primary aminegroups, using the immobilization wizard with the “Aim forimmobilization” option using Biacore™ Amine Coupling Kit (cat#BR-1000-50. A target immobilization of 5000 RU was specified. The wizarduses a 7 minute activation with a 1:1 mixture of 100 mM NHS(N-Hydroxysuccinimide) and 400 mM EDC(1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide), injects the ligand inseveral pulses to achieve the desired level, then deactivates theremaining surface with a 7 minute pulse of ethanolamine.

Anti-PCSK9 Fabs were captured on capture surface 2, and surface 1 wasused as a reference for kinetic studies of Fab:PCSK9 interactions. EachFab was captured by flowing a 500 ng/ml solution at 5 or 10 μl/min for1-1.5 minutes to reach a target R_(L) for an R max of 100-150 RU for thereaction. 5-10 concentrations of hPCSK9v5H is or mPCSK9v5H is antigenswere flowed across the surface at 30 μl/minute for 3-4 minutes. 15-60minutes dissociation time was allowed before regeneration of theAnti-Fab surface with a 30 second pulse of 10 mM glycine pH 2.0.

BiaEvaluation Software was used to evaluate the sensograms from themultiple concentration of PCSK9 antigen analyzed with each Fab, toestimate the kinetics constants of the Fab:PCSK9 interactions.

The kinetic constants were determined as follows:

TABLE 2 1B20 Fab hPCSK9v5His mPCSK9v5His k_(a) (1/Ms) 6.6E+04 ± 6.1E+031.41E+05 ± 1.2E+04  k_(d) (1/s) 4.8E−05 ± 7.4E−06 7.2E−05 ± 2.9E−06K_(A) (1/M) 1.5E+09 ± 3.0E+08 2.0E+09 ± 1.5E+08 K_(D) (M) 7.4E−10 ±1.6E−10 5.1E−10 ± 3.8E−11

EXAMPLE 7 Kinetic Evaluation of IgG:PCSK9 Interactions with SurfacePlasmon Resonance (“SPR”)

SPR measurements were performed using a Biacore™ (Pharmacia BiosensorAB, Uppsala, Sweden) 2000 system. Sensor chip CM5 and Amine Coupling Kitfor immobilization were from Biacore™.

A goat Anti-Human IgG (Caltag, catalog #H10700) was covalently coupledto surfaces 1 and 2 of a Sensor Chip CM5 via primary amine groups, usingthe immobilization wizard with the “Aim for immobilization” option usingBiacore™ Amine Coupling Kit (cat# BR-1000-50. A target immobilization of5000 RU was specified. The wizard uses a 7 minute activation with a 1:1mixture of 100 mM NHS (N-Hydroxysuccinimide) and 400 mM EDC(1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide), injects the ligand inseveral pulses to achieve the desired level, then deactivates theremaining surface with a 7 minute pulse of ethanolamine.

Anti-PCSK9 IgGs were captured on capture surface 2, and surface 1 wasused as a reference for kinetic studies of IgG:PCSK9 interactions. IgGwas captured by flowing a 10 nM solution at 10 μl/min for 1-1.5 minutesto reach a target R_(L) for an R max of 100-150 RU for the reaction.5-10 concentrations of hPCSK9v5H is or mPCSK9v5H is antigens were flowedacross the surface at 30 or 60 μl/minute for 4 minutes. 15-60 minutesdissociation time was allowed before regeneration of the Anti-IgGsurface with a 60 second pulse of 10 mM Glycine pH 1.7.

BiaEvaluation Software was used to evaluate the sensograms from themultiple concentration of PCSK9 antigen analyzed with each IgG, toestimate the kinetics constants of the IgG:PCSK9 interactions.

The kinetic constants were determined as follows:

TABLE 3 1B20 IgG hPCSK9v5His mPCSK9v5His k_(a) (1/Ms) 5.3E+04 ± 6.8E+038.9E+04 ± 3.2E+03 k_(d) (1/s) 4.3E−05 ± 4.5E−06 1.2E−04 ± 6.4E−06 K_(A)(1/M) 1.4E+09 ± 2.8E+08 7.4E+08 ± 4.2E+07 K_(D) (M) 8.9E−10 ± 1.6E−101.4E−09 ± 8.2E−11

EXAMPLE 8 PCSK9-LDLR TR-FRET Assay for 1B20

This assay is a variant of the one described in Fisher et al., 2007 J.Biol. Chem. 282:20502-20512. AlexaFluor647-labeled PCSK9 (finalconcentration 10 nM) was combined with varying amounts of 1B20 and tothis was added Eu(8044)-labeled LDLR ectodomain to a final concentrationof ˜4 nM (sufficient to give ˜20,000 counts at Fl₆₂₀ nM on the Rubystar)in 10 mM HEPES (pH 7.4), 150 mM NaCl, 0.1 mM CaCl₂, 0.05% (w/v) BSA in atotal volume of 50 μL using 96 well black Dynatech U bottom plates.After at least 90 minutes of equilibration, samples were read in aRubystar reader (BMG Corp.) using 20 flashes per well, a 50 usecintegration delay, and a 200 usec total integration time. Data wereexpressed as the ratio of (Fl₆₆₅/Fl₆₂₀×10000) and an IC₅₀ for 1B20 wasdetermined from the inflection point of a sigmoidal dose-response curveusing a standard four parameter fit.

FIG. 2 illustrates the activity of 1B20 in the PCSK9-LDLR interactionTR-FRET assay. Both the Fab and IgG of 1B20 are potent and inhibit thePCSK9-LDLR interaction fully.

EXAMPLE 9 Exopolar Assay Effects of Exogenous PCSK90N Cellular LDLUptake

On day 1, 30,000 HEK cells/well were plated in a 96 well polyD-lysinecoated plate. On day 2, the media was switched to no-serum containingDMEM media. On day 3, the media was removed and the cells were washedwith OptiMEM. Purified PCSK9 was added in 100 μl of DMEM mediacontaining LPDS and dI-LDL. The plates were incubated at 37° C. for 6.5hrs. The cells were washed quickly in TBS containing 2 mg/ml BSA; thenwashed in TBS-BSA for 2 minutes; and then washed twice (but quickly)with TBS. The cells were lysed in 100 μl RIPA buffer. Fluorescence wasthen measured in the plate using an Ex 520, Em 580 nm. The totalcellular protein in each well was measured using a BCA Protein Assay andthe fluorescence units were then normalized to total protein.

The Exopolar Assay is effective for characterizing variant effects onLDL uptake; see Table 4 below illustrating how the potencies of PCSK9mutants correlate with plasma LDL-cholesterol in the Exopolar Assay.

TABLE 4 EC-50 (nM) Mutation Gain/Loss LDL-C (mg/dI) Exopolar S127R Gain277 14 D374Y Gain 388 1.3 Wild-type 140 51 R46L Loss 116 78

Results: 1B20, both Fab and IgG, dose-dependently inhibited the effectsof both human and murine PCSK9 on LDL uptake; an effect which wasreproducibly observed. The amount of PCSK9 added to the cells was˜60-320 nM.

1B20 (Fab) comprises a light chain of SEQ ID NO: 1 (comprising a VL ofSEQ ID NO: 27) and a Fd chain of SEQ ID NO: 9 inclusive of linkers andtags (comprising a VH of SEQ ID NO: 11).

1B20 (IgG) comprises a light chain of SEQ ID NO: 26, and a heavy chaincomprising SEQ ID NO: 25.

FIGS. 3A-3D illustrate (i) 1B20 (Fab)'s dose-dependent inhibition ofmurine PCSK9-dependent loss of cellular LDL-uptake (FIG. 3A); (ii) 1B20(Fab)'s dose-dependent inhibition of human PCSK9-dependent loss ofcellular LDL-uptake (FIG. 3B); (iii) 1B20 (IgG)'s dose-dependentinhibition of murine PCSK9-dependent loss of cellular LDL-uptake (FIG.3C); and (iv) 1B20 (IgG)'s dose-dependent inhibition of humanPSCK9-dependent loss of cellular LDL-uptake (FIG. 3D).

1B20 clearly cross reacts with both human and mouse PCSK9. FIGS. 3A-3Dhave two controls: (i) a cell only control, showing the basal level ofcellular LDL uptake, and (ii) a PCSK9 (5 μg/ml) control which shows thelevel of PCSK9-dependent loss of LDL-uptake. The titration experimentswhich contain 1B20 and PCSK9 were done at a fixed concentration of PCSK9(5 μg/ml) and increasing concentrations of 1B20 shown in the graphs.

1B20 can inhibit the effect of PCSK9 on cellular LDL uptake. IC₅₀s for1B20 (Fab) are 152 nM (n=5) and 145 nM (n=5) for mouse and human PCSK9protein, respectively. IC₅₀s for 1B20 (IgG) are 13 nM and 22 nM formouse and human PCSK9 protein, respectively.

EXAMPLE 10 PCSK9 Cellular Uptake

The assay that follows was carried out according to the methods ofFisher et al., 2007 J. Biol. Chem. 282: 20502-12.

Cells treated with Alexa Fluor 647-labeled PCSK9 were imaged as follows.CHO cells were plated on poly-D-lysine-coated 96-well optical CVGsterile black plates (Nunc) at a density of 20,000 cells/well. Cellswere plated in F-12K medium (nutrient mixture, Kaighn's modification(1×)) (Invitrogen) containing 100 units of penicillin and 100 μg/mlstreptomycin sulfate and supplemented with 10% FBS. Plates wereincubated overnight at 37° C. and 5% CO₂. The following morning, themedium was removed and replaced with 100 μl of F-12K medium containing100 units of penicillin and 100 μg/ml streptomycin sulfate. After 18 h,the medium was removed. Purified PCSK9 protein was labeled with AlexaFluor 647 as described under “Experimental Procedures.” Alexa Fluor647-labeled PCSK9 (1, 5, or 20 μg/ml) was added in 50 μl of F-12K mediumcontaining 10% lipoprotein-deficient serum to the cells. The plates wereincubated at 37° C. for 4 h, and the cells were washed quickly withTris-buffered saline before imaging. To label cellular nuclei, Hoechst33342 at a final concentration of 0.1 μg/ml was added to each well. Theplates were run on an Opera imager (Evotec Technologies GmbH, Hamburg,Germany) with a x40 water immersion objective. Images were capturedusing excitation wavelengths of 405 nm for fluorescent nuclei and 635 nmfor Alexa Fluor 647-labeled PCSK9. For each well, 11 individual fieldscontaining >500 cells were captured for two emission wavelengths. Thedata were analyzed using a customized algorithm written using theAcapella language (Evotec Technologies GmbH). The algorithm identifiedand marked the nuclear and cytoplasmic areas of individual cells,followed by measurement of the total cytoplasmic intensity of the cell.The intensity was expressed in arbitrary fluorescent units.

For testing the 1B20 Ab, the identical procedure was used, with HEK293cells.

Results: FIG. 4 illustrates inhibition of PCSK9 internalization by the1B20 IgG.

EXAMPLE 11 In Vivo ASSAY

Whole IgG of human 1B20 was tested in vivo in mice and changes in thelevel of LDL cholesterol were monitored. The mice used in these studieswere (B6×B6-Tg(CETP) Ldlr^(tml)) F1 mice which are hemizygous for thetransgenic (Tg) expression of human CETP (which mice lack) as well asthe disruption of the LDL receptor (tml). These mice are particularlyuseful because of their human-like lipid profiles and LDL-rich nature.

Each mouse was bled twice, once at the beginning of the study toestablish individual baseline levels of LDL cholesterol (“pre”) and asecond time 3 hours later (“post”) to assess what changes took place inLDL levels after treatment. Each mouse received two IV doses ofDulbecco's PBS as a vehicle control, 1B20 IgG (0.5 mg), or 1B20 Fabfragments (0.5 mg) over the course of 3 hours. The 1B20 whole IgG wascentrifuged at 230,000×g to remove aggregates immediately prior toinjection.

In FIG. 5, the LDL levels for each mouse are represented by a set ofconnected symbols and the change in LDL (postbleed—prebleed) is shown asan average for each treatment group (Δ mg/dL). Treatment with PBS had noeffect on LDL measurements (−4 mg/dL, 5% reduction). In contrast, serumLDL was reduced 20% with 1B20 whole IgG (−19 mg/dL).

EXAMPLE 12 1B20 Rhesus PK/PD Study

To characterize pharmacokinetics, pharmacodynamics and target engagementof 1B20, a single dose IV study was conducted in male Rhesus monkeys at1, 3 and 10 mg/kg respectively (3.8-9.6 kg, n=3 per group). All Rhesusmonkeys used in the study were naïve to biologics.

Monkeys were given an IV bolus dose of 1B20 via the cephalic orsaphenous vein. Blood samples were collected from the saphenous/femoralvessel at designated time points post dosing and the resultingplasma/serum was stored at −70° C. until analysis.

The dosing solutions of 1B20 were prepared at 10 mg/mL (for 1 mg/kgdose) or 37.1 mg/mL (3 and 10 mg/kg dose) in 100 mM Histidine, 100 mMArginine, 6% sucrose, pH 6.0. The dosing solutions were stored at 4° C.and kept on wet ice during dosing.

The lipoprotein analysis of the serum samples were carried out asdescribed below. An anti-human IgG ELISA using commercially availablereagents was used to quantify 1B20 levels.

As shown in FIG. 7, 1B20 lowered LDL-C by ≧50% at all 3 doses tested and≧25% LDL-C lowering was observed for ≧8 days. The t_(1/2) of 1B20 (FIG.8) was 39 hr.

EXAMPLE 13 Lipoprotein Analysis of Plasma/Serum Samples from 1B20 RhesusPK/PD Study

To generate lipoprotein profiles, plasma or serum was fractionated bychromatography over Superose-6 size exclusion column (GE LifeSciences,Inc.). Total cholesterol levels in the column effluent were continuouslymeasured via in-line mixture with a commercially available enzymaticcolorimetric cholesterol detection reagent (Total Cholesterol E, WakoUSA) followed by downstream spectrophotometric detection of the reactionproducts at 600 nm absorbance. The first peak of cholesterol eluted fromthe column was attributed to VLDL, the second peak to LDL and the thirdto HDL; the area under each peak was calculated using software providedwith the HPLC. To calculate the cholesterol concentration for eachlipoprotein fraction, the ratio of the corresponding peak area to totalpeak area was multiplied by the total cholesterol concentration measuredin the sample.

EXAMPLE 14 Formulation

Monoclonal antibodies directed towards different therapeutic targets,including but not limited to mAb1 (that comprising a light chaincomprising SEQ ID NO: 26 and a heavy chain comprising SEQ ID NO: 25)were dialyzed into the appropriate formulations and concentrated to atarget concentration (50, 100, 125 or 150 mg/mL). Bulk solutions werethen dispensed into 3 mL glass vials for stability studies. Studiescarried out in liquid form were immediately placed on stability at 2-8°C., 25° C. and 37° C. Lyophilized samples were lyophilized in a labscale lyophilizer and the resulting lyophilized cake placed on stabilitystations at the same temperatures as the liquid samples.

Analytical methods included Size Exclusion Chromatography (SEC-HPLC) tomeasure aggregation and fragmentation, Dynamic Light Scattering (DLS) tomeasure particle size of concentrated samples, capillary SDS-PAGE tomeasure fragmentation and capillary iso-electric focusing (cIEF) tomeasure acidic variants formation.

The liquid stability of mAb1 (the PCSK9-specific antagonist referred toabove), as well as that of mAb2, mAb3 and mAb4 (alternate antibodies,two of which are in an IgG2m4 framework disclosed herein and one ofwhich is an IgG1, each one specific to a distinct targets) was enhancedwhen stored in a formulation of either 3% sucrose, 50 mM histidine, 50mM arginine, pH 6.0 or 6% sucrose, 100 mM histidine, 100 mM arginine, pH6.0 compared with other formulations tested, e.g., formulationscontaining sodium chloride, phosphate or varying lower concentrations ofsucrose, histidine and arginine. The lyophilized stability of mAb2 andmAb3 (mAb 1 not tested) was enhanced when stored in a pre-lyophilizationformulation of 3% sucrose, 50 mM histidine, 50 mM arginine, pH6.0 andremained stable after reconstitution with 0.5 times the original volume(0.5 ml; 0.5× original concentration of 1 ml), resulting in aformulation of approximately 6% sucrose, 100 mM histidine, 100 mMarginine, pH 6.0 and double protein concentration.

The lyophilized stability of mAb2 and mAb3 (mAb1 not tested) wasenhanced when stored in a lyophilized formulation containing 3% sucrose,50 mM histidine, 50 mM arginine, pH 6.0. Lyophilized samples were thenreconstituted with half of the original volume of water, resulting in aformulation of 6% sucrose, 10 mM histidine, 100 mM arginine, pH 6.0 anda protein concentration of 100 mg/mL. Both aggregation and fragmentationwere suppressed in the liquid and lyophilized formulations for all mAb'stested.

As illustrated in Table 5 below, the appearance of mAb1 after 1 month ofstorage at 45° C. was clear in the formulations of 3/50/50 and6/100/100. 3/100/0 and 2/25/25 appear slightly cloudy. The otherformulations tested appeared cloudy. A cloudy appearance can be anindication of visible aggregates being formed or of the solutionbeginning to separate into two phases.

TABLE 5 Appearance of various formulations after 1 month of storage at45° C. Sample ID* Temp (° C.) Appearance 10 His/150 NaCl 45 Cloudy6/100/100 45 Clear 6/100/100 PS80 45 Clear 3/50/50 pH 5.0 45 Cloudy,solid precipitate visible 3/50/50 45 Clear 3/100/0 45 Slightly cloudy2/25/25 45 Slightly cloudy *pH = 6.0 except where indicated otherwise.

Increased clipping of mAb1 was observed at pH 5.0 and in theHistidine/NaCl formulation (table 6).

TABLE 6 Changes in clipping events at non-reducing conditions after 3months of storage. Non-Reduced Difference from Time 0 in PercentResiduals (clipping) Formulation ID 2-8° C. 3M 25° C. 3M 37° C. 3M 10His/150 NaCl 0.1 0.3 5.2 6/100/100 0.15 0.3 1.1 6/100/100 PS80 0.1 0.41.6 3/50/50 pH 5.0 0.05 2.7 13.95 3/50/50 0.1 0.25 2.1 3/100/0 0.2 0.32.8 2/25/25 0.1 0.4 2.8 Highlighted cell indicate significant levels ofclipping.

The rate of aggregation of mAb1 at 37° C. is slowed when stored in aliquid formulation of 3% sucrose, 50 mM histidine, 50 mM arginine, pH6.0 or 6% sucrose, 100 mM histidine, 100 mM arginine, pH 6.0; see FIG.9.

Particle size of mAb1 in histidine/NaCl particle size increased muchfaster than any formulation containing a combination of sucrose,histidine and arginine. This indicates that the protein isself-associating and/or forming aggregates; see FIG. 10.

After 6 months of storage at 25° C. at 50 mg/mL, mAb2 contains the leastaggregation when stored in a formulation of 3% sucrose, 50 mM histidine,50 mM arginine (3/50/50) than other formulations tested; see FIG. 11.After the same amount of time at 25° C., the aggregation levels of mAb2in 3% sucrose, 50 mM histidine, 50 mM arginine, pH 6.0 (3/50/50) and 6%sucrose, 100 mM histidine, 100 mM arginine, pH 6.0 (6/100/100) areidentical; see FIG. 12.

mAb2 is stable for up to 6 months at 25° C. in 3% sucrose, 50 mMhistidine, 50 mM arginine, pH 6.0 at 50 mg/mL, 100 mg/mL and 150 mg/mL;see FIG. 13.

mAb3 is stable in 3/50/50 and 6/100/100 at 50 mg/mL for 6 months at 25°C. Fewer aggregates were observed in these formulations and in 2/25/25than in other liquid formulations tested; see FIG. 14.

Increased aggregation of mAb3 is observed with increasing concentrationin 3/50/50, but the rate of increase at 25° C. is minimal; see FIG. 15.

Minimal aggregation of mAb4 observed after storage at 25° C. for 12months in formulations of 3/50/50 or 2/25/25 compared with otherformulations tested; see FIGS. 16 and 17.

No aggregation was observed in a lyophilized formulation of 3% sucrose,50 mM histidine, 50 mM arginine, pH 6.0 at 25° C. in mAb2 (FIG. 18) ormAb3 (FIG. 19). Aggregation increased significantly in otherformulations tested.

EXAMPLE 15 Variants

Mutant 1B20 sequences were designed and libraries were generated andscreened for 1B20 derivatives. Library optimizations were conductedgenerally in accordance with U.S. Pat. No. 7,117,096. The libraries werethen screened and panned to identify variants with PCSK9-binding.Anti-PCSK9 antibody molecules were identified and are disclosed hereinas SEQ ID NOs: 45-96. The following table summarizes the Kd dataobtained exhibited by the antibodies from Biacore® analyses.

TABLE 7 Ab ID Comprising VH Kd (nM) A6 SEQ ID NO: 45 1.39 G3 SEQ ID NO:46 1.53 G5 SEQ ID NO: 47 1.65 B2 SEQ ID NO: 48 2.21 E2 SEQ ID NO: 492.62 G4 SEQ ID NO: 50 2.90 F4 SEQ ID NO: 51 3.14 B9 SEQ ID NO: 52 3.81C3 SEQ ID NO: 53 4.50 F2 SEQ ID NO: 54 1.01 F7 SEQ ID NO: 55 1.26 A7 SEQID NO: 56 1.28 G8 SEQ ID NO: 57 1.51 H4 SEQ ID NO: 58 1.52 D5 SEQ ID NO:59 1.60 D4 SEQ ID NO: 60 1.91 B4 SEQ ID NO: 61 2.01 H1 SEQ ID NO: 622.01 G2 SEQ ID NO: 63 2.11 A1 SEQ ID NO: 64 2.15 A4 SEQ ID NO: 65 2.15C2 SEQ ID NO: 66 2.25 H5 SEQ ID NO: 67 2.41 F6 SEQ ID NO: 68 2.68 B6 SEQID NO: 69 2.88 B1 SEQ ID NO: 70 3.73 F1 SEQ ID NO: 71 3.47 A8 SEQ ID NO:72 1.19 B3 SEQ ID NO: 73 1.21 F8 SEQ ID NO: 74 1.35 H8 SEQ ID NO: 751.35 B5 SEQ ID NO: 76 1.36 E1 SEQ ID NO: 77 1.36 E8 SEQ ID NO: 78 1.62C1 SEQ ID NO: 79 1.65 H3 SEQ ID NO: 80 1.89 A9 SEQ ID NO: 81 1.95 G7 SEQID NO: 82 2.05 C6 SEQ ID NO: 83 2.10 G6 SEQ ID NO: 84 2.20 E4 SEQ ID NO:85 2.38 F5 SEQ ID NO: 86 2.41 C7 SEQ ID NO: 87 2.53 E3 SEQ ID NO: 882.62 D3 SEQ ID NO: 89 2.68 D8 SEQ ID NO: 90 3.21 C8 SEQ ID NO: 91 3.73E5 SEQ ID NO: 92 4.22 B8 SEQ ID NO: 93 4.47 H7 SEQ ID NO: 94 4.90 A5 SEQID NO: 95 6.81 A3 SEQ ID NO: 96 7.93

Additional site-directed mutant variants of 1B20 were generated(mutations in the heavy chain) and are disclosed herein as SEQ ID NOs:102-107. Kds of site-directed mutant variants of 1B20 Fabs weredetermined using a Bio-Rad ProteOn; with affinity being measured againsthuman PCSK9-V5-His. The methodologies for measuring Fab affinities areessentially the same as previously described for Biacore®.

TABLE 8 Ab ID Comprising VH KD (nM) N59K SEQ ID NO: 102 0.013 N59Q SEQID NO: 103 0.117 N59R SEQ ID NO: 104 0.049 W101A SEQ ID NO: 105 1.37W101F SEQ ID NO: 106 1.12 W101Y SEQ ID NO: 107 0.780 * Amino acidnumbering begins with the first residue of FR1, immediately followingsignal peptide.

1. An isolated PCSK9-specific antagonist antibody or antigen-bindingfragment thereof which comprises: (a) a heavy chain variable regioncomprising CDR1, 2 and 3 domains; said CDR1 domain comprising the aminoacid sequence set forth in SEQ ID NO: 13; said CDR2 domain comprisingthe amino acid sequence set forth in SEQ ID NO: 15; and said CDR3 domaincomprising the amino acid sequence set forth in SEQ ID NO: 17; and (b) alight chain variable region comprising CDR1, 2 and 3 domains; said CDR1domain comprising the amino acid sequence set forth in SEQ ID NO: 3;said CDR2 domain comprising the amino acid sequence set forth in SEQ IDNO: 5, and said CDR3 domain comprising the amino acid sequence set forthin SEQ ID NO: 7; wherein said PCSK9-specific antagonist antibody orfragment antagonizes PCSK9-mediated inhibition of cellular LDL uptake.2. The PCSK9-specific antagonist antibody or antigen-binding fragmentthereof Of claim 1 wherein the CDR1, 2 and 3 domains are in a humangermline variable region in the respective CDR1, 2 and 3 regionsthereof.
 3. The PCSK9-specific antagonist antibody or antigen-bindingfragment thereof of claim 1 that binds to human PCSK9 with anequilibrium dissociation constant of less than 1200 nM,
 4. ThePCSK9-specific antagonist antibody or antigen-binding fragment thereofof claim 1 that antagonizes PCSK9-mediated inhibition of cellular LDLuptake at an IC₅₀ of less than 500 nM.
 5. The PCSK9-specific antagonistantibody or antigen-binding fragment thereof of claim 1 that antagonizesPCSK9-mediated inhibition of cellular uptake by at least 20%.
 6. ThePCSK9-specific antagonist antibody or antigen-binding fragment thereofof claim 1 which comprises a heavy chain variable region comprising theamino acid sequence set forth in SEQ ID NO: 11 and/or a light chainvariable region comprising the amino acid sequence set forth in SEQ IDNO:
 27. 7. The PCSK9-specific antagonist antibody or antigen-bindingfragtherit thereof of claim 1 which comprises a heavy chain havingconstant immunoglobulin sequence comprising the amino acid sequence setforth in SEQ ID NO: 24,
 8. An isolated PCSK9-specific antagonistantibody or antigen-binding fragment thereof which comprises: (a) alight chain comprising the amino acid sequence set forth in SEQ ID NO:26; and (b) a heavy chain comprising the amino acid sequence set forthin SEQ ID NO: 25; wherein said PCSK9-specific antagonist antibody orfragment antagonizes PCSK9-mediated inhibition of cellular LDL uptake.9. An isolated PCSK9-specific antagonist antibody or antigen-bindingfragment thereof which comprises: (a) a heavy chain variable regioncomprising CDR1, 2 and 3 domains; said CDR1 domain comprising the aminoacid sequence set forth in SEQ ID NO: 37; said CDR2 domain comprisingthe amino acid sequence set forth in SEQ ID NO: 38; and said CDR3 domaincomprising the amino acid sequence set forth in SEQ ID NO: 39; and alight chain variable region comprising COR1, 2 and 3 domains; said CDR1domain comprising the amino acid sequence set forth in SEQ ID NO: 40;said CDR2 domain comprising the amino acid sequence set forth in SEQ IDNO: 41, and said CDR3 domain comprising the amino acid sequence setforth in SEQ ID NO: 42; (b) a heavy chain variable region comprisingCDR1, 2 and 3 domains; said CDR1 domain compriing the amino acidsequence set forth in SEQ ID NO: 37; said CDR2 domain comprising theamino acid sequence set forth in SEQ ID NO: 97; and said CDR3 domaincomprising the amino acid sequence set forth in SEQ ID NO: 98; and alight chain variable region comprising CDR1, 2 and 3 domains; said CDR1domain comprising the amino acid sequence set forth in SEQ ID NO: 99;said CDR2 domain comprising the amino acid sequence set forth in SEQ IDNO: 100, and said CDR3 domain comprising the amino acid sequence setforth in SEQ ID NO: 101; or (c) a heavy chain variable region comprisingthe amino acid sequence set forth in any one of SEQ ID NOs: 45-96 or102-107 and a light chain variable region comprising the amino acidsequence set forth in SEQ ID NO:27; wherein said PCSK9-specificantagonist antibody or fragment antagonizes PCSK9-mediated inhibition ofcellular LDL uptake.
 10. A composition comprising the PCSK9-specificantagonist antibody or antigen-binding fragment thereof of claim 1 and apharmaceutically acceptable carrier.
 11. A composition comprising thePCSK9-specific antagonist antibody or antigen-binding fragment thereofof claim 1 and a further therapeutic drug.
 12. The Composition of claim11 wherein the further therapeutic drug is a cholesterol absorptioninhibitor or a cholesterol synthesis inhibitor.
 13. A compositioncomprising the PCSK9-specific antagonist antibody or antigen-bindingfragment thereof of claim 9 and a pharmaceutically acceptable carrier.14. An isolated host cell or population of host cells in vitro or insitu comprising a PCSK9-specific antagonist antibody or antigen-bindingfragment thereof of claim
 1. 15. The host cell of claim 14 which is aPichia celIor a Chinese hamster ovary cell.
 16. An isolated host cell orpopulation of host cells in vitro or in situ comprising a PCSK9-specificantagonist antibody or antigen-binding fragment thereof of claim
 9. 17.The host cell of claim 16 which is a Pichia cell or a Chinese hamsterovary cell.
 18. A method for lowering plasma low density lipoproteinlevel in a patient which comprises administering the PCSK9-specificantagonist antibody or antigen-binding fragment thereof of claim 1 tothe patient.
 19. The method of claim 18 wherein the patient isadministered a further therapeutic drug.
 20. The method of claim 19wherein the further therapeutic drug is acholesterol absorptioninhibitor or a cholesterol synthesis inhibitor.
 21. A method fortreating a medical condition mediated by PCSK9 activity in a patientcomprising administering a therapeutically effective amount ofPCSK9-specific antagonist antibody or antigen-binding fragment thereofof claim 1 to the patient.
 22. The method of claim 21 wherein themedical condition is hypercholesterolemia, coronary heart disease,metabolic syndrome or acute coronary syndrome.